Antioxidant inflammation modulators: oleanolic acid derivatives with saturation in the C-ring

ABSTRACT

This invention provides, but is not limited to, novel oleanolic acid derivatives having the formula: wherein the variables are defined herein. Also provided are pharmaceutical compositions, kits and articles of manufacture comprising such compounds, methods and intermediates useful for making the compounds, and methods of using the compounds and compositions.

The present application is a continuation application of applicationSer. No. 12/426,737, filed Apr. 20, 2009 now U.S. Pat. No. 7,915,402,which claims the priority of U.S. Provisional Application Nos.61/046,332, filed Apr. 18, 2008, and 61/111,333, filed Nov. 4, 2008, theentire contents of each of which are incorporated herein by reference intheir entirety without disclaimer.

BACKGROUND OF THE INVENTION

I. Field of the Invention

The present invention relates generally to the fields of biology andmedicine. More particularly, it concerns compounds and methods for thetreatment and prevention of diseases such as those associated withoxidative stress and inflammation.

II. Description of Related Art

Many serious and intractable human diseases are associated withdysregulation of inflammatory processes, including diseases such ascancer, atherosclerosis, and diabetes, which were not traditionallyviewed as inflammatory conditions. Similarly, autoimmune diseases suchas rheumatoid arthritis, lupus, psoriasis, and multiple sclerosisinvolve inappropriate and chronic activation of inflammatory processesin affected tissues, arising from dysfunction of self vs. non-selfrecognition and response mechanisms in the immune system. Inneurodegenerative diseases such as Alzheimer's and Parkinson's diseases,neural damage is correlated with activation of microglia and elevatedlevels of pro-inflammatory proteins such as inducible nitric oxidesynthase (iNOS).

One aspect of inflammation is the production of inflammatoryprostaglandins such as prostaglandin E, whose precursors are produced bythe enzyme cyclo-oxygenase (COX-2). High levels of COX-2 are found ininflamed tissues. Consequently, inhibition of COX-2 is known to reducemany symptoms of inflammation and a number of importantanti-inflammatory drugs (e.g., ibuprofen and celecoxib) act byinhibiting COX-2 activity. Recent research, however, has demonstratedthat a class of cyclopentenone prostaglandins (e.g., 15-deoxyprostaglandin J2, a.k.a. PGJ2) plays a role in stimulating theorchestrated resolution of inflammation. COX-2 is also associated withthe production of cyclopentenone prostaglandins. Consequently,inhibition of COX-2 may interfere with the full resolution ofinflammation, potentially promoting the persistence of activated immunecells in tissues and leading to chronic, “smoldering” inflammation. Thiseffect may be responsible for the increased incidence of cardiovasculardisease in patients using selective COX-2 inhibitors for long periods oftime. Corticosteroids, another important class of anti-inflammatorydrugs, have many undesirable side effects and frequently are notsuitable for chronic use. Newer protein-based drugs, such as anti-TNFmonoclonal antibodies, have proven to be effective for the treatment ofcertain autoimmune diseases such as rheumatoid arthritis. However, thesecompounds must be administered by injection, are not effective in allpatients, and may have severe side effects. In many severe forms ofinflammation (e.g., sepsis, acute pancreatitis), existing drugs areineffective. In addition, currently available drugs typically do nothave significant antioxidant properties, and are not effective inreducing oxidative stress associated with excessive production ofreactive oxygen species and related molecules such as peroxynitrite.Accordingly, there is a pressing need for improved therapeutics withantioxidant and anti-inflammatory properties.

A series of synthetic triterpenoid analogs of oleanolic acid have beenshown to be inhibitors of cellular inflammatory processes, such as theinduction by IFN-γ of inducible nitric oxide synthase (iNOS) and ofCOX-2 in mouse macrophages. See Honda et al. (2000a); Honda et al.(2000b), and Honda et al. (2002), which are all incorporated herein byreference. For example, one of these,2-cyano-3,12-dioxooleane-1,9(11)-dien-28-oic acid methyl ester(CDDO-Me), is currently in clinical trials for a variety of disordersrelated to inflammation, including cancer and diabetic nephropathy. Thepharmacology of these molecules is complex, as they have been shown toaffect the function of multiple protein targets and thereby modulate thefunction of several important cellular signaling pathways related tooxidative stress, cell cycle control, and inflammation (e.g.,Dinkova-Kostova et al., Ahmad et al., 2006; Ahmad et al., 2008; Liby etal.,). Given that the biological activity profiles of the knownoleanolic acid derivatives vary, and in view of the wide variety ofdiseases that may be treated with compounds having potent antioxidantand anti-inflammatory effects, it is desirable to synthesize newcandidates for the treatment or prevention of disease.

SUMMARY OF THE INVENTION

The present disclosure provides new compounds with antioxidant andanti-inflammatory properties, methods for their manufacture, and methodsfor their use. Compounds covered by the generic or specific formulasbelow or specifically named are referred to as “compounds of theinvention,” “compounds of the present disclosure,” “the presentoleanolic acid derivatives” in the present application.

In some aspects, the disclosure provides compounds of the formula:

wherein:

-   -   Y is cyano, heteroaryl_((C≦12)), substituted        heteroaryl_((C≦12)), or —C(O)R_(a), further        -   wherein R_(a) is:            -   hydrogen, hydroxy, halo, amino, hydroxyamino, azido,                silyl or mercapto;            -   alkyl_((C≦12)), alkenyl_((C≦12)), alkynyl_((C≦12)),                aryl_((C≦12)), aralkyl_((C≦12)), heteroaryl_((C≦12)),                heteroaralkyl_((C≦12)), alkoxy_((C≦12)),                alkenyl-oxy_((C≦12)), alkynyloxy_((C≦12)),                aryloxy_((C≦12)), aralkoxy_((C≦12)),                heteroaryloxy_((C≦12)), heteroaralkoxy_((C≦12)),                acyloxy_((C≦12)), alkylamino_((C≦12)),                dialkylamino_((C≦12)), alkoxyamino_((C≦12)),                alkenylamino_((C≦12)), alkynylamino_((C≦12)),                arylamino_((C≦12)), aralkylamino_((C≦12)),                heteroarylamino_((C≦12)), heteroaralkyl-amino_((C≦12)),                alkylsulfonylamino_((C≦12)), amido_((C≦12)),                alkyl-thio_((C≦12)), alkenylthio_((C≦12)),                alkynylthio_((C≦12)), arylthio_((C≦12)),                aralkylthio_((C≦12)), heteroarylthio_((C≦12)),                heteroaralkylthio_((C≦12)), acylthio_((C≦12)),                alkylammonium_((C≦12)), alkylsulfonium_((C≦12)),                alkylsilyl_((C≦12)), or a substituted version of any of                these groups; or            -   R_(a) comprises a nitrogen atom that is also attached to                carbon atom 13 and R_(d) to form:

-   -   -   -   -   wherein R_(d) is alkyl_((C≦8)), alkenyl_((C≦8)),                    aryl_((C≦8)), aralkyl_((C≦8)), heteroaryl_((C≦8)),                    heteroaralkyl_((C≦8)), or a substituted version of                    any of these groups;

    -   Z is a single or double bond, —O— or —NR_(e)—, wherein R_(e) is        hydrogen, hydroxy, alkyl_((C≦8)) or alkoxy_((C≦8));

    -   X is OR_(b), NR_(b)R_(c), or SR_(b), wherein R_(b) and R_(c) are        each independently:        -   hydrogen or hydroxy;        -   alkyl_((C≦8)), aryl_((C≦8)), aralkyl_((C≦8)), acyl_((C≦8)),            alkoxy_((C≦8)), aryloxy_((C≦8)), acyloxy_((C≦8)),            alkylamino_((C≦8)), arylamino_((C≦8)), amido_((C≦8)), or a            substituted version of any of these groups; or        -   a substituent convertible in vivo to hydrogen;        -   provided that R_(b) is absent when the atom to which it is            bound is part of a double bond, further provided that when            R_(b) is absent the atom to which it is bound is part of a            double bond;

    -   R₁ is:        -   hydrogen, cyano, hydroxy, halo or amino; or        -   alkyl_((C≦8)), alkenyl_((C≦8)), alkynyl_((C≦8)),            aryl_((C≦8)), aralkyl_((C≦8)), heteroaryl_((C≦8)),            heteroaralkyl_((C≦8)), acyl_((C≦8)), alkoxy_((C≦8)),            aryloxy_((C≦8)), acyloxy_((C≦8)), alkylamino_((C≦8)),            arylamino_((C≦8)), amido_((C≦8)), or a substituted version            of any of these groups;

    -   R₂ is:        -   cyano, hydroxy, halo or amino; or        -   fluoroalkyl_((C≦8)), alkenyl_((C≦8)), alkynyl_((C≦8)),            aryl_((C≦8)), heteroaryl_((C≦8)), acyl_((C≦8)),            alkoxy_((C≦8)), aryloxy_((C≦8)), acyloxy_((C≦8)),            alkylamino_((C≦8)), arylamino_((C≦8)), amido_((C≦8)), or a            substituted version of any of these groups;

    -   R₃ is:        -   absent or hydrogen;        -   alkyl_((C≦8)), aryl_((C≦8)), aralkyl_((C≦8)), acyl_((C≦8)),            or a substituted version of any of these groups; or        -   a substituent convertible in vivo to hydrogen;        -   provided that R₃ is absent when the oxygen atom to which it            is bound is part of a double bond, further provided that            when R₃ is absent the oxygen atom to which it is bound is            part of a double bond;

    -   R₄ and R₅ are each independently alkyl_((C≦8)) or substituted        alkyl_((C≦8));

    -   R₆ is hydrogen, hydroxy or oxo;

    -   R₇ is hydrogen or hydroxy; and

    -   R₈, R₉, R₁₀ and R₁₁ are each independently hydrogen, hydroxy,        alkyl_((C≦8)), substituted alkyl_((C≦8)), alkoxy_((C≦8)) or        substituted alkoxy_((C≦8));        or pharmaceutically acceptable salts, esters, hydrates,        solvates, tautomers, prodrugs, or optical isomers thereof.

In some embodiments, the compound is further defined as:

wherein:

-   -   Y is cyano, or —C(O)R_(a), further wherein:        -   R_(a) is:            -   hydrogen, hydroxy, halo, amino, hydroxyamino, azido or                mercapto; or            -   alkyl_((C≦12)), alkenyl_((C≦12)), alkynyl_((C≦12)),                aryl_((C≦12)), aralkyl_((C≦12)), heteroaryl_((C≦12)),                heteroaralkyl_((C≦12)), alkoxy_((C≦12)),                alkenyl-oxy_((C≦12)), alkynyloxy_((C≦12)),                aryloxy_((C≦12)), aralkoxy_((C≦12)),                heteroaryloxy_((C≦12)), heteroaralkoxy_((C≦12)),                acyloxy_((C≦12)), alkylamino_((C≦12)),                dialkylamino_((C≦12)), alkoxyamino_((C≦12)),                alkenylamino_((C≦12)), alkynylamino_((C≦12)),                arylamino_((C≦12)), aralkylamino_((C≦12)),                heteroarylamino_((C≦12)), heteroaralkyl-amino_((C≦12)),                alkylsulfonylamino_((C≦12)), amido_((C≦12)),                alkyl-thio_((C≦12)), alkenylthio_((C≦12)),                alkynylthio_((C≦12)), arylthio_((C≦12)),                aralkylthio_((C≦12)), heteroarylthio_((C≦12)),                heteroaralkylthio_((C≦12)), acylthio_((C≦12)),                alkylammonium_((C≦12)), alkylsulfonium_((C≦12)),                alkylsilyl_((C≦12)), or a substituted version of any of                these groups;    -   X is OR_(b), NR_(b)R_(c), or SR_(b), wherein R_(b) and R_(c) are        each independently:        -   hydrogen or hydroxy;        -   alkyl_((C≦8)), aryl_((C≦8)), aralkyl_((C≦8)), acyl_((C≦8)),            alkoxy_((C≦8)), aryloxy_((C≦8)), acyloxy_((C≦8)),            alkylamino_((C≦8)), arylamino_((C≦8)), amido_((C≦8)), or        -   a substituted version of any of these groups; or        -   a substituent convertible in vivo to hydrogen;        -   provided that R_(b) is absent when the atom to which it is            bound is part of a double bond, further provided that when            R_(b) is absent the atom to which it is bound is part of a            double bond;    -   R₁ is:        -   hydrogen, cyano, hydroxy, halo or amino; or        -   alkyl_((C≦8)), alkenyl_((C≦8)), alkynyl_((C≦8)),            aryl_((C≦8)), aralkyl_((C≦8)), heteroaryl_((C≦8)),            heteroaralkyl_((C≦8)), acyl_((C≦8)), alkoxy_((C≦8)),            aryloxy_((C≦8)), acyloxy_((C≦8)), alkylamino_((C≦8)),            arylamino_((C≦8)), amido_((C≦8)), or a substituted version            of any of these groups;    -   R₂ is:        -   cyano, hydroxy, halo or amino; or        -   fluoroalkyl_((C≦8)), alkenyl_((C≦8)), alkynyl_((C≦8)),            aryl_((C≦8)), heteroaryl_((C≦8)), acyl_((C≦8)),            alkoxy_((C≦8)), aryloxy_((C≦8)), acyloxy_((C≦8)),            alkylamino_((C≦8)), arylamino_((C≦8)), amido_((C≦8)), or a            substituted version of any of these groups;    -   R₃ is:        -   absent or hydrogen;        -   alkyl_((C≦8)), aryl_((C≦8)), aralkyl_((C≦8)), acyl_((C≦8)),            or a substituted version of any of these groups; or        -   a substituent convertible in vivo to hydrogen;        -   provided that R₃ is absent when the oxygen atom to which it            is bound is part of a double bond, further provided that            when R₃ is absent the oxygen atom to which it is bound is            part of a double bond;    -   R₄ and R₅ are each independently alkyl_((C≦8)) or substituted        alkyl_((C≦8)); and    -   R₆ and R₇ are each independently hydrogen or hydroxy;        or pharmaceutically acceptable salts, esters, hydrates,        solvates, tautomers, prodrugs, or optical isomers thereof.

In some embodiments, the compound is further defined as:

wherein:

-   -   Y is cyano or —C(O)R_(a), further wherein:        -   R_(a) is:            -   hydrogen, hydroxy, halo, amino, azido, mercapto or                silyl; or            -   alkyl_((C≦12)), alkenyl_((C≦12)), alkynyl_((C≦12)),                aryl_((C≦12)), aralkyl_((C≦12)), heteroaryl_((C≦12)),                heteroaralkyl_((C≦12)), alkoxy_((C≦12)),                alkenyl-oxy_((C≦12)), alkynyloxy_((C≦12)),                aryloxy_((C≦12)), aralkoxy_((C≦12)),                heteroaryloxy_((C≦12)), heteroaralkoxy_((C≦12)),                acyloxy_((C≦12)), alkylamino_((C≦12)),                alkoxyamino_((C≦12)), dialkylamino_((C≦12)),                alkenylamino_((C≦12)), alkynylamino_((C≦12)),                arylamino_((C≦12)), aralkylamino_((C≦12)),                heteroarylamino_((C≦12)), heteroaralkyl-amino_((C≦12)),                amido_((C≦12)), or a substituted version of any of these                groups;    -   X is OR_(b), NR_(b)R_(c), or SR_(b), wherein R_(b) and R_(c) are        each independently:        -   hydrogen or hydroxy;        -   alkyl_((C≦8)), aryl_((C≦8)), aralkyl_((C≦8)), acyl_((C≦8)),            alkoxy_((C≦8)), aryloxy_((C≦8)), acyloxy_((C≦8)),            alkylamino_((C≦8)), arylamino_((C≦8)), amido_((C≦8)), or a            substituted version of any of these groups; or        -   a substituent convertible in vivo to hydrogen;        -   provided that R_(b) is absent when the atom to which it is            bound is part of a double bond, further provided that when            R_(b) is absent the atom to which it is bound is part of a            double bond;    -   R₁ is:        -   hydrogen, cyano, hydroxy, halo or amino; or        -   alkyl_((C≦8)), alkenyl_((C≦8)), alkynyl_((C≦8)),            aryl_((C≦8)), aralkyl_((C≦8)), heteroaryl_((C≦8)),            heteroaralkyl_((C≦8)), acyl_((C≦8)), alkoxy_((C≦8)),            aryloxy_((C≦8)), acyloxy_((C≦8)), alkylamino_((C≦8)),            arylamino_((C≦8)), amido_((C≦8)), or a substituted version            of any of these groups;    -   R₂ is:        -   cyano, hydroxy, halo or amino; or        -   fluoroalkyl_((C≦8)), alkenyl_((C≦8)), alkynyl_((C≦8)),            aryl_((C≦8)), heteroaryl_((C≦8)), acyl_((C≦8)),            alkoxy_((C≦8)), aryloxy_((C≦8)), acyloxy_((C≦8)),            alkylamino_((C≦8)) arylamino_((C≦8)), amido_((C≦8)), or a            substituted version of any of these groups; and    -   R₄ is alkyl_((C≦8)) or substituted alkyl_((C≦8));        or pharmaceutically acceptable salts, esters, hydrates,        solvates, tautomers, prodrugs, or optical isomers thereof.

In some embodiments, the compound is further defined as:

wherein:

-   -   R_(a) is:        -   hydrogen, hydroxy, halo, or amino; or        -   alkyl_((C≦8)), alkenyl_((C≦8)), alkynyl_((C≦8)),            aryl_((C≦8)), aralkyl_((C≦8)), heteroaryl_((C≦8)),            heteroaralkyl_((C≦8)), alkoxy_((C≦8)), alkenyloxy_((C≦8)),            alkynyloxy_((C≦8)), aryloxy_((C≦8)), aralkoxy_((C≦8)),            heteroaryloxy_((C≦8)), heteroaralkoxy_((C≦8)),            acyloxy_((C≦8)), alkylamino_((C≦8)), alkoxyamino_((C≦8)),            dialkylamino_((C≦8)), alkenylamino_((C≦8)),            alkynylamino_((C≦8)), arylamino_((C≦8)),            aralkyl-amino_((C≦8)), heteroarylamino_((C≦8)),            heteroaralkylamino_((C≦8)), amido_((C≦8)), or a substituted            version of any of these groups;    -   X is OR_(b) or NR_(b)R_(c), wherein R_(b) and R_(c) are each        independently:        -   hydrogen or hydroxy;        -   alkyl_((C≦8)), aryl_((C≦8)), aralkyl_((C≦8)), acyl_((C≦8)),            alkoxy_((C≦8)), aryloxy_((C≦8)), acyloxy_((C≦8)),            alkylamino_((C≦8)), arylamino_((C≦8)), amido_((C≦8)), or a            substituted version of any of these groups; or        -   a substituent convertible in vivo to hydrogen;        -   provided that R_(b) is absent when the atom to which it is            bound is part of a double bond, further provided that when            R_(b) is absent the atom to which it is bound is part of a            double bond; and    -   R₂ is:        -   cyano, hydroxy, halo or amino; or        -   fluoroalkyl_((C≦8)), alkenyl_((C≦8)), alkynyl_((C≦8)),            aryl_((C≦8)), heteroaryl_((C≦8)), acyl_((C≦8)),            alkoxy_((C≦8)), aryloxy_((C≦8)), acyloxy_((C≦8)),            alkylamino_((C≦8)), arylamino_((C≦8)), amido_((C≦8)), or a            substituted version of any of these groups;            or pharmaceutically acceptable salts, esters, hydrates,            solvates, tautomers, prodrugs, or optical isomers thereof.

In some embodiments, the compound is further defined as:

wherein:

-   -   R_(a) is:        -   hydrogen, hydroxy, halo, or amino; or        -   alkyl_((C≦8)), alkenyl_((C≦8)), alkynyl_((C≦8)),            aryl_((C≦8)), aralkyl_((C≦8)), heteroaryl_((C≦8)),            heteroaralkyl_((C≦8)), alkoxy_((C≦8)), alkenyloxy_((C≦8)),            alkynyloxy_((C≦8)), aryloxy_((C≦8)), aralkoxy_((C≦8)),            heteroaryloxy_((C≦8)), heteroaralkoxy_((C≦8)),            acyloxy_((C≦8)), alkylamino_((C≦8)), alkoxyamino_((C≦8)),            dialkylamino_((C≦8)), alkenylamino_((C≦8)),            alkynylamino_((C≦8)), arylamino_((C≦8)),            aralkyl-amino_((C≦8)), heteroarylamino_((C≦8)),            heteroaralkylamino_((C≦8)), amido_((C≦8)), or a substituted            version of any of these groups; and    -   R₂ is:        -   cyano or fluoro; or        -   fluoroalkyl_((C≦5)), alkenyl_((C≦5)), alkynyl_((C≦5)),            heteroaryl_((C≦5)), acyl_((C≦5)), acyloxy_((C≦5)),            amido_((C≦5)), or a substituted version of any of these            groups;            or pharmaceutically acceptable salts, esters, hydrates,            solvates, tautomers, prodrugs, or optical isomers thereof.

In some embodiments, the compound is further defined as:

wherein R_(a) is:

-   -   hydrogen, hydroxy, halo or amino; or    -   alkyl_((C≦8)), alkenyl_((C≦8)), alkynyl_((C≦8)), aryl_((C≦8)),        aralkyl_((C≦8)), heteroaryl_((C≦8)), heteroaralkyl_((C≦8)),        alkoxy_((C≦8)), alkenyloxy_((C≦8)), alkynyloxy_((C≦8)),        aryloxy_((C≦8)), aralkoxy_((C≦8)), heteroaryloxy_((C≦8)),        heteroaralkoxy_((C≦8)), acyloxy_((C≦8)), alkylamino_((C≦8)),        alkoxyamino_((C≦8)), dialkylamino_((C≦8)),        alkenyl-amino_((C≦8)), alkynylamino_((C≦8)), arylamino_((C≦8)),        aralkylamino_((C≦8)), heteroarylamino_((C≦8)),        heteroaralkylamino_((C≦8)), amido_((C≦8)), or a substituted        version of any of these groups;        or pharmaceutically acceptable salts, esters, hydrates,        solvates, tautomers, prodrugs, or optical isomers thereof.

In some embodiments, the compound is further defined as:

wherein R_(a) is alkoxy_((C1-4)), alkylamino_((C1-4)),alkoxyamino_((C1-4)), dialkylamino_((C2-4)), or a substituted version ofany of these groups; or pharmaceutically acceptable salts, esters,hydrates, solvates, tautomers, prodrugs, or optical isomers thereof.

In some embodiments, the compound is further defined as:

wherein R_(a) is alkyl_((C1-4)) or aralkoxy_((C7-8)) or a substitutedversion of either of these groups; or pharmaceutically acceptable salts,esters, hydrates, solvates, tautomers, prodrugs, or optical isomersthereof.

In some embodiments, the compound is further defined as:

wherein R_(a) is hydrogen, hydroxy, amino, dimethylamino, methyl,methoxy, methoxyamino, benzyloxy, or 2,2,2-trifluoroethylamino; orpharmaceutically acceptable salts, hydrates, solvates, tautomers, oroptical isomers thereof.

In some embodiments, the compound is further defined as:

wherein R_(a) is hydrogen, hydroxy, amino, methoxy, or2,2,2-trifluoroethylamino; or pharmaceutically acceptable salts,hydrates, solvates, tautomers, or optical isomers thereof.

In some embodiments, the compound is further defined as:

wherein R_(d) is alkyl_((C≦8)), alkenyl_((C≦8)), aryl_((C≦8)),aralkyl_((C≦8)), heteroaryl_((C≦8)), heteroaralkyl_((C≦8)), or asubstituted version of any of these groups; or pharmaceuticallyacceptable salts, hydrates, solvates, tautomers, or optical isomersthereof.

In some embodiments, the compound is further defined as:

wherein Y is heteroaryl_((C≦8)) or a substituted heteroaryl_((C≦8)); ora pharmaceutically acceptable salts, hydrates, solvates, tautomers, oroptical isomers thereof.

In some embodiments, the compound is further defined as:

wherein:

-   -   wherein Y is cyano or —C(O)R_(a), further wherein:        -   R_(a) is:            -   hydrogen, hydroxy, halo, amino, azido, mercapto or                silyl; or            -   alkyl_((C≦12)), alkenyl_((C≦12)), alkynyl_((C≦12)),                aryl_((C≦12)), aralkyl_((C≦12)), heteroaryl_((C≦12)),                heteroaralkyl_((C≦12)), alkoxy_((C≦12)),                alkenyl-oxy_((C≦12)), alkynyloxy_((C≦12)),                aryloxy_((C≦12)), aralkoxy_((C≦12)),                heteroaryloxy_((C≦12)), heteroaralkoxy_((C≦12)),                acyloxy_((C≦12)), alkylamino_((C≦12)),                alkoxyamino_((C≦12)), dialkylamino_((C≦12)),                alkenylamino_((C≦12)), alkynylamino_((C≦12)),                arylamino_((C≦12)), aralkylamino_((C≦12)),                heteroarylamino_((C≦12)), heteroaralkyl-amino_((C≦12)),                amido_((C≦12)), or a substituted version of any of these                groups;    -   R₂ is:        -   cyano, hydroxy, halo or amino; or        -   fluoroalkyl_((C≦8)), alkenyl_((C≦8)), alkynyl_((C≦8)),            aryl_((C≦8)), heteroaryl_((C≦8)), acyl_((C≦8)),            alkoxy_((C≦8)), aryloxy_((C≦8)), acyloxy_((C≦8)),            alkylamino_((C≦8)), arylamino_((C≦8)), amido_((C≦8)), or a            substituted version of any of these groups;    -   R₃ is:        -   absent or hydrogen;        -   alkyl_((C≦8)), aryl_((C≦8)), aralkyl_((C≦8)), acyl_((C≦8)),            or a substituted version of any of these groups; or        -   a substituent convertible in vivo to hydrogen;        -   provided that R₃ is absent when the oxygen atom to which it            is bound is part of a double bond, further provided that            when R₃ is absent the oxygen atom to which it is bound is            part of a double bond; and    -   R₄ is alkyl_((C≦8)) or substituted alkyl_((C≦8));        or pharmaceutically acceptable salts, esters, hydrates,        solvates, tautomers, prodrugs, or optical isomers thereof.

In some embodiments, the compound is further defined as:

wherein:

-   -   Y is cyano or —C(O)R_(a), wherein        -   R_(a) is:            -   hydrogen, hydroxy, halo, amino, hydroxyamino, azido or                mercapto; or            -   alkyl_((C≦12)), alkenyl_((C≦12)), alkynyl_((C≦12)),                aryl_((C≦12)), aralkyl_((C≦12)), heteroaryl_((C≦12)),                heteroaralkyl_((C≦12)), alkoxy_((C≦12)),                alkenyl-oxy_((C≦12)), alkynyloxy_((C≦12)),                aryloxy_((C≦12)), aralkoxy_((C≦12)),                heteroaryloxy_((C≦12)), heteroaralkoxy_((C≦12)),                acyloxy_((C≦12)), alkylamino_((C≦12)),                alkoxyamino_((C≦12)), dialkylamino_((C≦12)),                alkenylamino_((C≦12)), alkynylamino_((C≦12)),                arylamino_((C≦12)), aralkylamino_((C≦12)),                heteroarylamino_((C≦12)), heteroaralkyl-amino_((C≦12)),                alkylsulfonylamino_((C≦12)), amido_((C≦12)), or a                substituted version of any of these groups;    -   R₂ is:        -   cyano, hydroxy, halo or amino; or        -   fluoroalkyl_((C≦8)), alkenyl_((C≦8)), alkynyl_((C≦8)),            aryl_((C≦8)), heteroaryl_((C≦8)), acyl_((C≦8)),            alkoxy_((C≦8)), aryloxy_((C≦8)), acyloxy_((C≦8)),            alkylamino_((C≦8)), arylamino_((C≦8)), amido_((C≦8)), or a            substituted version of any of these groups; and    -   R₃ is:        -   hydrogen;        -   alkyl_((C≦8)), aryl_((C≦8)), aralkyl_((C≦8)), acyl_((C≦8)),            or a substituted version of any of these groups; or        -   a substituent convertible in vivo to hydrogen;            or pharmaceutically acceptable salts, esters, hydrates,            solvates, tautomers, prodrugs, or optical isomers thereof.

In some embodiments, the compound is further defined as:

wherein:

-   -   X is OR_(b), NR_(b)R_(c), or SR_(b), wherein R_(b) and R_(c) are        each independently:        -   hydrogen;        -   alkyl_((C≦8)), aryl_((C≦8)), aralkyl_((C≦8)), acyl_((C≦8)),            or a substituted version of any of these groups; or        -   a substituent convertible in vivo to hydrogen;        -   provided that R_(b) is absent when the atom to which it is            bound is part of a double bond, further provided that when            R_(b) is absent the atom to which it is bound is part of a            double bond;    -   R₁ is:        -   hydrogen, cyano, hydroxy, halo or amino; or        -   alkyl_((C≦8)), alkenyl_((C≦8)), alkynyl_((C≦8)),            aryl_((C≦8)), aralkyl_((C≦8)), heteroaryl_((C≦8)),            heteroaralkyl_((C≦8)), acyl_((C≦8)), alkoxy_((C≦8)),            aryloxy_((C≦8)), acyloxy_((C≦8)), alkylamino_((C≦8)),            arylamino_((C≦8)), amido_((C≦8)), or a substituted version            of any of these groups;    -   R₂ is:        -   cyano, hydroxy, halo or amino; or        -   fluoroalkyl_((C≦8)), alkenyl_((C≦8)), alkynyl_((C≦8)),            aryl_((C≦8)), heteroaryl_((C≦8)), acyl_((C≦8)),            alkoxy_((C≦8)), aryloxy_((C≦8)), acyloxy_((C≦8)),            alkylamino_((C≦8)) arylamino_((C≦8)), amido_((C≦8)), or a            substituted version of any of these groups; and    -   R₄ is alkyl_((C≦8)) or substituted alkyl_((C≦8));        or pharmaceutically acceptable salts, esters, hydrates,        solvates, tautomers, prodrugs, or optical isomers thereof.

In some embodiments, the compound is further defined as:

wherein:

-   -   X is OR_(b) or NR_(b)R_(c), wherein R_(b) and R_(c) are each        independently:        -   hydrogen;        -   alkyl_((C≦8)), aryl_((C≦8)), aralkyl_((C≦8)), acyl_((C≦8)),            or a substituted version of any of these groups; or        -   a substituent convertible in vivo to hydrogen;        -   provided that R_(b) is absent when the atom to which it is            bound is part of a double bond, further provided that when            R_(b) is absent the atom to which it is bound is part of a            double bond; and    -   R₂ is:        -   cyano, hydroxy, halo or amino; or        -   fluoroalkyl_((C≦8)), alkenyl_((C≦8)), alkynyl_((C≦8)),            aryl_((C≦8)), heteroaryl_((C≦8)), acyl_((C≦8)),            alkoxy_((C≦8)), aryloxy_((C≦8)), acyloxy_((C≦8)),            alkylamino_((C≦8)), arylamino_((C≦8)), amido_((C≦8)), or a            substituted version of any of these groups;            or pharmaceutically acceptable salts, esters, hydrates,            solvates, tautomers, prodrugs, or optical isomers thereof.

In some embodiments, the compound is further defined as:

wherein:

-   -   R_(a) is:        -   hydrogen, hydroxy, halo, amino, azido, mercapto or silyl; or        -   alkyl_((C≦12)), alkenyl_((C≦12)), alkynyl_((C≦12)),            aryl_((C≦12)), aralkyl_((C≦12)), heteroaryl_((C≦12)),            heteroaralkyl_((C≦12)), alkoxy_((C≦12)),            alkenyloxy_((C≦12)), alkynyloxy_((C≦12)), aryloxy_((C≦12)),            aralkoxy_((C≦12)), heteroaryloxy_((C≦12)),            heteroaralkoxy_((C≦12)), acyloxy_((C≦12)),            alkylamino_((C≦12)), alkoxy-amino_((C≦12)),            dialkylamino_((C≦12)), alkenylamino_((C≦12)),            alkynyl-amino_((C≦12)), arylamino_((C≦12)),            aralkylamino_((C≦12)), heteroaryl-amino_((C≦12)),            heteroaralkylamino_((C≦12)), amido_((C≦12)), or a            substituted version of any of these groups;    -   R₁ is:        -   hydrogen, cyano, hydroxy, halo or amino; or        -   alkyl_((C≦8)), alkenyl_((C≦8)), alkynyl_((C≦8)),            aryl_((C≦8)), aralkyl_((C≦8)), heteroaryl_((C≦8)),            heteroaralkyl_((C≦8)), acyl_((C≦8)), alkoxy_((C≦8)),            aryloxy_((C≦8)), acyloxy_((C≦8)), alkylamino_((C≦8)),            arylamino_((C≦8)), amido_((C≦8)), or a substituted version            of any of these groups;    -   R₂ is:        -   cyano, hydroxy, halo or amino; or        -   fluoroalkyl_((C≦8)), alkenyl_((C≦8)), alkynyl_((C≦8)),            aryl_((C≦8)), heteroaryl_((C≦8)), acyl_((C≦8)),            alkoxy_((C≦8)), aryloxy_((C≦8)), acyloxy_((C≦8)),            alkylamino_((C≦8)) arylamino_((C≦8)), amido_((C≦8)), or a            substituted version of any of these groups; and            or pharmaceutically acceptable salts, esters, hydrates,            solvates, tautomers, prodrugs, or optical isomers thereof.

In some embodiments, the compound is further defined as:

wherein R_(a) is:

-   -   hydrogen, hydroxy, halo or amino; or    -   alkyl_((C≦6)), aryl_((C≦8)), aralkyl_((C≦8)),        heteroaryl_((C≦8)), alkoxy_((C≦6)), aryloxy_((C≦8)),        aralkoxy_((C≦8)), alkylamino_((C≦6)), alkoxyamino_((C≦6)),        alkoxyamino_((C≦6)), dialkylamino_((C≦6)), arylamino_((C≦8)),        aralkylamino_((C≦8)), heteroarylamino_((C≦6)),        heteroarylamino_((C≦8)), amido_((C≦6)), or a substituted version        of any of these groups;        or pharmaceutically acceptable salts, esters, hydrates,        solvates, tautomers, prodrugs, or optical isomers thereof.

In some aspects, the disclosure provides compounds of the formula:

wherein:

-   -   Y is cyano or —C(O)R_(a), wherein        -   R_(a) is:            -   hydrogen, hydroxy, halo, amino, hydroxyamino, azido or                mercapto; or            -   alkyl_((C≦12)), alkenyl_((C≦12)), alkynyl_((C≦12)),                aryl_((C≦12)), aralkyl_((C≦12)), heteroaryl_((C≦12)),                heteroaralkyl_((C≦12)), alkoxy_((C≦12)),                alkenyl-oxy_((C≦12)), alkynyloxy_((C≦12)),                aryloxy_((C≦12)), aralkoxy_((C≦12)),                heteroaryloxy_((C≦12)), heteroaralkoxy_((C≦12)),                acyloxy_((C≦12)), alkylamino_((C≦12)),                dialkylamino_((C≦12)), alkoxyamino_((C≦12)),                alkenylamino_((C≦12)), alkynylamino_((C≦12)),                arylamino_((C≦12)), aralkylamino_((C≦12)),                heteroarylamino_((C≦12)), heteroaralkyl-amino_((C≦12)),                alkylsulfonylamino_((C≦12)), amido_((C≦12)),                alkyl-thio_((C≦12)), alkenylthio_((C≦12)),                alkynylthio_((C≦12)), arylthio_((C≦12)),                aralkylthio_((C≦12)), heteroarylthio_((C≦12)),                heteroaralkylthio_((C≦12)), acylthio_((C≦12)),                alkylammonium_((C≦12)), alkylsulfonium_((C≦12)),                alkylsilyl_((C≦12)), or a substituted version of any of                these groups;    -   X is OR_(b), NR_(b)R_(c), or SR_(b), wherein R_(b) and R_(c) are        each independently:        -   hydrogen;        -   alkyl_((C≦8)), aryl_((C≦8)), aralkyl_((C≦8)), acyl_((C≦8)),            or a substituted version of any of these groups; or        -   provided that R_(b) is absent when the atom to which it is            bound is part of a double bond, further provided that when            R_(b) is absent the atom to which it is bound is part of a            double bond; and    -   R₁ is:        -   hydrogen, cyano, hydroxy, halo or amino; or        -   alkyl_((C≦8)), alkenyl_((C≦8)), alkynyl_((C≦8)),            aryl_((C≦8)), aralkyl_((C≦8)), heteroaryl_((C≦8)),            heteroaralkyl_((C≦8)), acyl_((C≦8)), alkoxy_((C≦8)),            aryloxy_((C≦8)), acyloxy_((C≦8)), alkylamino_((C≦8)),            arylamino_((C≦8)), amido_((C≦8)), or a substituted version            of any of these groups;            or salts, esters, hydrates, solvates, tautomers, or optical            isomers thereof.

In some aspects, the disclosure provides compounds of the formula:

wherein:

-   -   Y is cyano or —C(O)R_(a), wherein        -   R_(a) is:            -   hydrogen, hydroxy, halo, amino, hydroxyamino, azido or                mercapto; or            -   alkyl_((C≦12)), alkenyl_((C≦12)), alkynyl_((C≦12)),                aryl_((C≦12)), aralkyl_((C≦12)), heteroaryl_((C≦12)),                heteroaralkyl_((C≦12)), alkoxy_((C≦12)),                alkenyl-oxy_((C≦12)), alkynyloxy_((C≦12)),                aryloxy_((C≦12)), aralkoxy_((C≦12)),                heteroaryloxy_((C≦12)), heteroaralkoxy_((C≦12)),                acyloxy_((C≦12)), alkylamino_((C≦12)),                dialkylamino_((C≦12)), alkoxyamino_((C≦12)),                alkenylamino_((C≦12)), alkynylamino_((C≦12)),                arylamino_((C≦12)), aralkylamino_((C≦12)),                heteroarylamino_((C≦12)), heteroaralkyl-amino_((C≦12)),                alkylsulfonylamino_((C≦12)), amido_((C≦12)),                alkyl-thio_((C≦12)), alkenylthio_((C≦12)),                alkynylthio_((C≦12)), arylthio_((C≦12)),                aralkylthio_((C≦12)), heteroarylthio_((C≦12)),                heteroaralkylthio_((C≦12)), acylthio_((C≦12)),                alkylammonium_((C≦12)), alkylsulfonium_((C≦12)),                alkylsilyl_((C≦12)), or a substituted version of any of                these groups;    -   X is OR_(b), NR_(b)R_(c), or SR_(b), wherein R_(b) and R_(c) are        each independently:        -   hydrogen;        -   alkyl_((C≦8)), aryl_((C≦8)), aralkyl_((C≦8)), acyl_((C≦8)),            or a substituted version of any of these groups; or        -   provided that R_(b) is absent when the atom to which it is            bound is part of a double bond, further provided that when            R_(b) is absent the atom to which it is bound is part of a            double bond; and    -   R₁ is:        -   hydrogen, cyano, hydroxy, halo or amino; or        -   alkyl_((C≦8)), alkenyl_((C≦8)), alkynyl_((C≦8)),            aryl_((C≦8)), aralkyl_((C≦8)), heteroaryl_((C≦8)),            heteroaralkyl_((C≦8)), acyl_((C≦8)), alkoxy_((C≦8)),            aryloxy_((C≦8)), acyloxy_((C≦8)), alkylamino_((C≦8)),            arylamino_((C≦8)), amido_((C≦8)), or a substituted version            of any of these groups;    -   R′ is hydroxy, alkoxy_((C≦12)), substituted alkoxy_((C≦12)),        aryloxy_((C≦12)), substituted aryloxy_((C≦12)),        aralkoxy_((C≦12)), substituted aralkoxy_((C≦12)),        acyloxy_((C≦12)), or substituted acyloxy_((C≦12));        or salts, esters, hydrates, solvates, tautomers, or optical        isomers thereof.

In a variation of each of the above embodiments containing a Z group, Zcan be a single bond, —O—, or —NH—. In a variation of each of the aboveembodiments containing an X group, X can be OR_(b). In some variations,R_(b) is absent. In other variations, R_(b) is hydrogen. In othervariations, X can be NR_(b). In some variations, R_(b) can be hydroxy.In a variation of each of the above embodiments containing a Y group, Ycan be cyano or —C(O)R_(a). In some variations, R_(a) can be hydroxy. Insome variations, R_(a) can be alkoxy_((C≦6)), aryloxy_((C≦8)),aralkyloxy_((C≦8)), or a substituted version of any of these groups. Insome of these variations, R_(a) can be alkoxy_((C2-6)). In some of thesevariations, R_(a) can be alkoxy_((C1-5)) or substituted alkoxy_((C1-5)).In some of these variations, R_(a) can be alkoxy_((C2-4)) or substitutedalkoxy_((C2-4)). In some of these variations, R_(a) can bealkoxy_((C1-2)) or substituted alkoxy_((C1-4)). In some of thesevariations, R_(a) can be alkoxy_((C1-2)) or substituted alkoxy_((C1-2)).For example, R_(a) can be methoxy. In some variations, R_(a) can beamino. In some variations, R_(a) can be alkylamino_((C1-6)),alkoxyamino_((C1-6)), arylamino_((C1-8)), aralkylamino_((C1-8)),dialkylamino_((C2-8)), or a substituted version of any of these groups.In some of these variations, R_(a) can be alkylamino_((C2-6)) orsubstituted alkylamino_((C2-6)). In some of these variations, R_(a) canbe alkylamino_((C3-6)). In some variations, R_(a) can bealkylamino_((C1-5)), dialkylamino_((C2-6)), or substituted version ofeither of these groups. In some variations, R_(a) can bealkylamino_((C2-4)), dialkylamino_((C2-5)), or substituted version ofeither of these groups. In some variations, R_(a) can bealkylamino_((C1-4)) or substituted alkylamino_((C1-4)). In somevariations, R_(a) can be alkylamino_((C1-3)). In some of thesevariations, R_(a) can be methylamino or ethylamino. In some of thesevariations, R_(a) can be substituted alkylamino_((C1-3)). For example,R_(a) can be 2,2,2-trifluoroethylamino. In some of these variations,R_(a) can be alkyl_((C1-5)), aryl_((C≦8)), aralkyl_((C≦8)),heteroaralkyl_((C≦8)), or a substituted version of any of these groups.In some of these variations, R_(a) can be heteroaryl_((C1-8)) orsubstituted heteroaryl_((C1-8)). For example, wherein R_(a) can beimidazolyl. In some of these variations, R_(a) can be —H.

In a variation of each of the above embodiments containing an R₁ group,R₁ can be —H, —OH or —F. For example, R₁ can be —H. In a variation ofeach of the above embodiments containing an R₂ group, R₂ can be —CN. Insome variations, R₂ can be a substituted acyl_((C1-3)), such as—C(═O)NHS(═O)₂CH₃. In some variations, R₂ is fluoroalkyl_((C≦8)). Forexample, R₂ can be —CF₃. In other variations, R₂ is notfluoroalkyl_((C≦8)).

In a variation of each of the above embodiments containing an R₃ group,R₃ can be hydrogen or acetyl. In another variation, R₃ can be absent. Ina variation of each of the above embodiments containing an R₄ group, R₄can be methyl or hydroxymethyl. In a variation of each of the aboveembodiments containing an R₆, R₇, R₈, or R₉ group, R₆, R₇, R₈, or R₉ canindependently be hydrogen. In a variation of each of the aboveembodiments containing an R₁₀ or R₁₁ group, R₁₀ or R₁₁ can independentlybe methyl. In a variation of each of the above embodiments containing anR′ group, R′ can be acetyloxy or hydroxy.

In a variation of each of the above embodiments containing an R_(d)group, R_(d) can be alkyl_((C1-5)), aryl_((C≦8)), aralkyl_((C≦8)),heteroaralkyl_((C≦8)), or a substituted version of any of these groups.In some variations, R_(d) can be alkyl_((C1-4)) or a substituted versionthereof. In some variations, R_(d) can be alkyl_((C1-3)) or asubstituted version thereof. In some variations, R_(d) can bealkyl_((C1-2)) or a substituted version thereof.

Non-limiting examples of compounds provided by this invention includethe compounds according to the formulas shown below, as well as orpharmaceutically acceptable salts thereof. In certain embodiments, thesecompounds are substantially free from other optical isomers thereof

Examples of specific compounds provided by the present disclosureinclude:

-   (4aS,6aR,6bR,8aR,12aR,14aR,14bS)-methyl    11-cyano-2,2,6a,6b,9,9,12a-heptamethyl-10,14-dioxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,12a,12b,13,14,14a,14b-icosahydropicene-4a-carboxylate,-   (4aS,6aR,6bR,8aR,12aR,14aR,14bS)-methyl    11-cyano-10-hydroxy-2,2,6a,6b,9,9,12a-heptamethyl-14-oxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,12,12a,12b,13,14,14a,14b-icosahydropicene-4a-carboxylate,-   (4aS,6aR,6bR,8aR,12aR,14aR,14bS)-11-cyano-2,2,6a,6b,9,9,12a-heptamethyl-10,14-dioxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,12a,12b,13,14,14a,14b-icosahydropicene-4a-carboxylic    acid,-   (4aR,6aR,6bR,8aS,12aS,12bR,14bR)-4,4,6a,6b,11,11,14b-heptamethyl-3,13-dioxo-3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14,14a,14b-icosahydropicene-2,8a-dicarbonitrile,-   (4aS,6aR,6bR,8aR,12aR,14aR,14bS)-11-cyano-2,2,6a,6b,9,9,12a-heptamethyl-10,14-dioxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,12a,12b,13,14,14a,14b-icosahydropicene-4a-carboxamide,-   (4aR,6aR,6bR,8aS,12aS,12bR,14bR)-8a-formyl-4,4,6a,6b,11,11,14b-heptamethyl-3,13-dioxo-3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14,14a,14b-icosahydropicene-2-carbonitrile,-   (4aS,6aR,6bR,8aR,12aR,14R,14aR,14bS)-methyl    11-cyano-14-hydroxy-2,2,6a,6b,9,9,12a-heptamethyl-10-oxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,12a,12b,13,14,14a,14b-icosahydropicene-4a-carboxylate,-   (4aS,6aR,6bR,8aR,12aR,14aR,14bS)-11-cyano-2,2,6a,6b,9,9,12a-heptamethyl-10,14-dioxo-N-(2,2,2-trifluoroethyl)-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,12a,12b,13,14,14a,14b-icosahydropicene-4a-carboxamide,-   (4aS,6aR,6bR,8aR,12aR,12bR,14R,14aR,14bS)-2,2,2-trifluoroethyl    11-cyano-14-hydroxy-2,2,6a,6b,9,9,12a-heptamethyl-10-oxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,12a,12b,13,14,14a,14b-icosahydropicene-4a-carboxylate,-   (4aS,6aR,6bR,8aR,12aR,12bR,14aR,14bS)-11-cyano-2,2,6a,6b,9,9,12a-heptamethyl-10,14-dioxo-N-(2,2,2-trifluoroethyl)-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,12a,12b,13,14,14a,14b-icosahydropicene-4a-carboxamide,-   (4aS,6aR,6bR,8aR,12aR,12bR,14aR,14bS)-N′-acetyl-11-cyano-2,2,6a,6b,9,9,12a-heptamethyl-10,14-dioxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,12a,12b,13,14,14a,14b-icosahydropicene-4a-carbohydrazide,-   (4aS,6aR,6bR,8aR,12aR,12bR,14aR,14bS)-11-cyano-2,2,6a,6b,9,9,12a-heptamethyl-10,14-dioxo-N-(2,2,2-trifluoroethyl)docosahydropicene-4a-carboxamide,-   (4aR,6aR,6bR,8aS,12aS,12bR,14aR,14bR)-4,4,6a,6b,11,11,14b-heptamethyl-3,13-dioxo-8a-(2H-tetrazol-5-yl)-3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14,14a,14b-icosahydropicene-2-carbonitrile,-   (4aR,6aR,6bR,8aS,12aS,12bR,14aR,14bR)-4,4,6a,6b,11,11,14b-heptamethyl-8a-(2-methyl-2H-tetrazol-5-yl)-3,13-dioxo-3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14,14a,14b-icosahydropicene-2-carbonitrile,-   (4aR,6aR,6bR,8aS,12aS,12bR,14aR,14bR)-4,4,6a,6b,11,11,14b-heptamethyl-8a-(5-methyl-1,3,4-oxadiazol-2-yl)-3,13-dioxo-3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14,14a,14b-icosahydropicene-2-carbonitrile,    and-   (4aR,6aR,6bR,8aS,12aS,12bR,14aR,14bR)-4,4,6a,6b,11,11,14b-heptamethyl-8a-(5-methyl-1,3,4-thiadiazol-2-yl)-3,13-dioxo-3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14,14a,14b-icosahydropicene-2-carbonitrile,-   (4aR,6aR,6bR,8aS,12aS,12bR,14aR,14bR)-8a-acetyl-4,4,6a,6b,11,11,14b-heptamethyl-3,13-dioxo-3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14,14a,14b-icosahydropicene-2-carbonitrile,-   (4aS,6aR,6bR,8aR,12aR,12bR,14aR,14bS)-benzyl    11-cyano-2,2,6a,6b,9,9,12a-heptamethyl-10,14-dioxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,12a,12b,13,14,14a,14b-icosahydropicene-4a-carboxylate,-   (4aS,6aR,6bR,8aR,12aR,12bR,14aR,14bS)-11-cyano-N,N,2,2,6a,6b,9,9,12a-nonamethyl-10,14-dioxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,12a,12b,13,14,14a,14b-icosahydropicene-4a-carboxamide,-   (4aS,6aR,6bR,8aR,12aR,12bR,14aR,14bS)-11-cyano-N-methoxy-2,2,6a,6b,9,9,12a-heptamethyl-10,14-dioxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,12a,12b,13,14,14a,14b-icosahydropicene-4a-carboxamide-   (4aS,6aR,6bR,8aR,12aR,12bR,14aR,14bS)-methyl    11-cyano-14-(hydroxyimino)-2,2,6a,6b,9,9,12a-heptamethyl-10-oxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,12a,12b,13,14,14a,14b-icosahydropicene-4a-carboxylate,-   (4aR,6aR,6bS,8aS,12aR,15aR,15bR)-methyl    2-cyano-14-hydroxy-4,4,6a,6b,11,11,15b-heptamethyl-3-oxo-3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,14,15,15a,15b-icosahydrodinaphtho[1,2-b:2′,1′-d]oxepine-8a-carboxylate,    and-   (4aR,6aR,6bS,8aS,12aR,12bR,15aR,15bR)-methyl    2-cyano-4,4,6a,6b,11,11,15b-heptamethyl-3,14-dioxo-4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14,15,15a,15b-icosahydro-3H-dinaphtho[1,2-b:2′,1′-d]azepine-8a-carboxylate.

In some embodiments, compounds of the present disclosure are in the formof pharmaceutically acceptable salts. In other embodiments, compounds ofthe present disclosure are not in the form of a pharmaceuticallyacceptable salts. In some embodiments, compounds of the presentdisclosure are in the form of a hydrate. In other embodiments, compoundsof the present disclosure are not in the form of a hydrate. In someembodiments, compounds of the present disclosure are in the form of asolvate. In other embodiments, compounds of the present disclosure arenot in the form of a solvate.

In some embodiments, compounds of the present disclosure can be estersof the above formulas. The ester may, for example, result from acondensation reaction between a hydroxy group of the formula and thecarboxylic acid group of biotin. In other embodiments, compounds of thepresent disclosure are not an ester.

In some embodiments, the compounds of the present disclosure can bepresent as a mixture of stereoisomers. In other embodiments, thecompounds of the present disclosure are present as single stereoisomers.

In some embodiments, compounds of the present disclosure may beinhibitors of IFN-γ-induced nitrous oxide (NO) production inmacrophages, for example, having an IC₅₀ value of less than 0.2 μM.

Other general aspects of the present disclosure contemplate apharmaceutical composition comprising as an active ingredient a compoundof the present disclosure and a pharmaceutically acceptable carrier. Thecomposition may, for example, be adapted for administration by a routeselected from the group consisting of orally, intraadiposally,intraarterially, intraarticularly, intracranially, intradermally,intralesionally, intramuscularly, intranasally, intraocularally,intrapericardially, intraperitoneally, intrapleurally,intraprostaticaly, intrarectally, intrathecally, intratracheally,intratumorally, intraumbilically, intravaginally, intravenously,intravesicularlly, intravitreally, liposomally, locally, mucosally,orally, parenterally, rectally, subconjunctival, subcutaneously,sublingually, topically, transbuccally, transdermally, vaginally, incrèmes, in lipid compositions, via a catheter, via a lavage, viacontinuous infusion, via infusion, via inhalation, via injection, vialocal delivery, via localized perfusion, bathing target cells directly,or any combination thereof. In particular embodiments, the compositionmay be formulated for oral delivery. In particular embodiments, thecomposition is formulated as a hard or soft capsule, a tablet, a syrup,a suspension, a wafer, or an elixir. In certain embodiments, the softcapsule is a gelatin capsule. Certain compositions may comprise aprotective coating, such as those compositions formulated for oraldelivery. Certain compositions further comprise an agent that delaysabsorption, such as those compositions formulated for oral delivery.Certain compositions may further comprise an agent that enhancessolubility or dispersibility, such as those compositions formulated fororal delivery. Certain compositions may comprise a compound of thepresent disclosure, wherein the compound is dispersed in a liposome, anoil in water emulsion or a water in oil emulsion.

Yet another general aspect of the present disclosure contemplates atherapeutic method comprising administering a pharmaceutically effectivecompound of the present disclosure to a subject. The subject may, forexample, be a human. These or any other methods of the presentdisclosure may further comprise identifying a subject in need oftreatment.

Another method of the present disclosure contemplates a method oftreating cancer in a subject, comprising administering to the subject apharmaceutically effective amount of a compound of the presentdisclosure. The cancer may be any type of cancer, such as a carcinoma,sarcoma, lymphoma, leukemia, melanoma, mesothelioma, multiple myeloma,or seminoma. Other types of cancers include cancer of the bladder,blood, bone, brain, breast, central nervous system, colon, endometrium,esophagus, genitourinary tract, head, larynx, liver, lung, neck, ovary,pancreas, prostate, spleen, small intestine, large intestine, stomach,or testicle. In these or any other methods, the subject may be aprimate. In these or any other methods, the subject may be a human. Thisor any other method may further comprise identifying a subject in needof treatment. The subject may have a family or patient history ofcancer. In certain embodiments, the subject has symptoms of cancer. Thecompounds of the invention may be administered via any method describedherein, such as locally. In certain embodiments, the compound isadministered by direct intratumoral injection or by injection into tumorvasculature. In certain embodiments, the compounds may be administeredsystemically. The compounds may be administered intravenously,intra-arterially, intramuscularly, intraperitoneally, subcutaneously ororally, in certain embodiments.

In certain embodiments regarding methods of treating cancer in asubject, comprising administering to the subject a pharmaceuticallyeffective amount of a compound of the present disclosure, thepharmaceutically effective amount is 0.1-1000 mg/kg. In certainembodiments, the pharmaceutically effective amount is administered in asingle dose per day. In certain embodiments, the pharmaceuticallyeffective amount is administered in two or more doses per day. Thecompound may be administered by contacting a tumor cell during ex vivopurging, for example. The method of treatment may comprise any one ormore of the following: a) inducing cytotoxicity in a tumor cell; b)killing a tumor cell; c) inducing apoptosis in a tumor cell; d) inducingdifferentiation in a tumor cell; or e) inhibiting growth in a tumorcell. The tumor cell may be any type of tumor cell, such as a leukemiacell. Other types of cells include, for example, a bladder cancer cell,a breast cancer cell, a lung cancer cell, a colon cancer cell, aprostate cancer cell, a liver cancer cell, a pancreatic cancer cell, astomach cancer cell, a testicular cancer cell, a brain cancer cell, anovarian cancer cell, a lymphatic cancer cell, a skin cancer cell, abrain cancer cell, a bone cancer cell, or a soft tissue cancer cell.

Combination treatment therapy is also contemplated by the presentdisclosure. For example, regarding methods of treating cancer in asubject, comprising administering to the subject a pharmaceuticallyeffective amount of a compound of the present disclosure, the method mayfurther comprise a treatment selected from the group consisting ofadministering a pharmaceutically effective amount of a second drug,radiotherapy, gene therapy, and surgery. Such methods may furthercomprise (1) contacting a tumor cell with the compound prior tocontacting the tumor cell with the second drug, (2) contacting a tumorcell with the second drug prior to contacting the tumor cell with thecompound, or (3) contacting a tumor cell with the compound and thesecond drug at the same time. The second drug may, in certainembodiments, be an antibiotic, anti-inflammatory, anti-neoplastic,anti-proliferative, anti-viral, immunomodulatory, or immunosuppressive.The second drug may be an alkylating agent, androgen receptor modulator,cytoskeletal disruptor, estrogen receptor modulator, histone-deacetylaseinhibitor, HMG-CoA reductase inhibitor, prenyl-protein transferaseinhibitor, retinoid receptor modulator, topoisomerase inhibitor, ortyrosine kinase inhibitor. In certain embodiments, the second drug is5-azacitidine, 5-fluorouracil, 9-cis-retinoic acid, actinomycin D,alitretinoin, all-trans-retinoic acid, annamycin, axitinib, belinostat,bevacizumab, bexarotene, bosutinib, busulfan, capecitabine, carboplatin,carmustine, CD437, cediranib, cetuximab, chlorambucil, cisplatin,cyclophosphamide, cytarabine, dacarbazine, dasatinib, daunorubicin,decitabine, docetaxel, dolastatin-10, doxifluridine, doxorubicin,doxorubicin, epirubicin, erlotinib, etoposide, etoposide, gefitinib,gemcitabine, gemtuzumab ozogamicin, hexamethylmelamine, idarubicin,ifosfamide, imatinib, irinotecan, isotretinoin, ixabepilone, lapatinib,LBH589, lomustine, mechlorethamine, melphalan, mercaptopurine,methotrexate, mitomycin, mitoxantrone, MS-275, neratinib, nilotinib,nitrosourea, oxaliplatin, paclitaxel, plicamycin, procarbazine,semaxanib, semustine, sodium butyrate, sodium phenylacetate,streptozotocin, suberoylanilide hydroxamic acid, sunitinib, tamoxifen,teniposide, thiopeta, tioguanine, topotecan, TRAIL, trastuzumab,tretinoin, trichostatin A, valproic acid, valrubicin, vandetanib,vinblastine, vincristine, vindesine, or vinorelbine.

Methods of treating or preventing a disease with an inflammatorycomponent in a subject, comprising administering to the subject apharmaceutically effective amount of a compound of the presentdisclosure are also contemplated. The disease may be, for example, lupusor rheumatoid arthritis. The disease may be an inflammatory boweldisease, such as Crohn's disease or ulcerative colitis. The disease withan inflammatory component may be a cardiovascular disease. The diseasewith an inflammatory component may be diabetes, such as type 1 or type 2diabetes. Compounds of the present disclosure may also be used to treatcomplications associated with diabetes. Such complications arewell-known in the art and include, for example, obesity, hypertension,atherosclerosis, coronary heart disease, stroke, peripheral vasculardisease, hypertension, nephropathy, neuropathy, myonecrosis, retinopathyand metabolic syndrome (syndrome X). The disease with an inflammatorycomponent may be a skin disease, such as psoriasis, acne, or atopicdermatitis. Administration of a compound of the present disclosure intreatment methods of such skin diseases may be, for example, topical ororal.

The disease with an inflammatory component may be metabolic syndrome(syndrome X). A patient having this syndrome is characterized as havingthree or more symptoms selected from the following group of fivesymptoms: (1) abdominal obesity; (2) hypertriglyceridemia; (3) lowhigh-density lipoprotein cholesterol (HDL); (4) high blood pressure; and(5) elevated fasting glucose, which may be in the range characteristicof Type 2 diabetes if the patient is also diabetic. Each of thesesymptoms is defined in the Third Report of the National CholesterolEducation Program Expert Panel on Detection, Evaluation and Treatment ofHigh Blood Cholesterol in Adults (Adult Treatment Panel III, or ATPIII), National Institutes of Health, 2001, NIH Publication No. 01-3670,incorporated herein by reference. Patients with metabolic syndrome,whether or not they have or develop overt diabetes mellitus, have anincreased risk of developing the macrovascular and microvascularcomplications that are listed above that occur with type 2 diabetes,such as atherosclerosis and coronary heart disease.

Another general method of the present disclosure entails a method oftreating or preventing a cardiovascular disease in a subject, comprisingadministering to the subject a pharmaceutically effective amount of acompound of the present disclosure. The cardiovascular disease may be,for example, atherosclerosis, cardiomyopathy, congenital heart disease,congestive heart failure, myocarditis, rheumatic heart disease, valvedisease, coronary artery disease, endocarditis, or myocardialinfarction. Combination therapy is also contemplated for such methods.For example, such methods may further comprise administering apharmaceutically effective amount of a second drug. The second drug maybe, for example, a cholesterol lowering drug, an anti-hyperlipidemic, acalcium channel blocker, an anti-hypertensive, or an HMG-CoA reductaseinhibitor. Non-limiting examples of second drugs include amlodipine,aspirin, ezetimibe, felodipine, lacidipine, lercanidipine, nicardipine,nifedipine, nimodipine, nisoldipine or nitrendipine. Other non-limitingexamples of second drugs include atenolol, bucindolol, carvedilol,clonidine, doxazosin, indoramin, labetalol, methyldopa, metoprolol,nadolol, oxprenolol, phenoxybenzamine, phentolamine, pindolol, prazosin,propranolol, terazosin, timolol or tolazoline. The second drug may be,for example, a statin, such as atorvastatin, cerivastatin, fluvastatin,lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin orsimvastatin.

Methods of treating or preventing a neurodegenerative disease in asubject, comprising administering to the subject a pharmaceuticallyeffective amount of a compound of the present disclosure are alsocontemplated. The neurodegenerative disease may, for example, beselected from the group consisting of Parkinson's disease, Alzheimer'sdisease, multiple sclerosis (MS), Huntington's disease and amyotrophiclateral sclerosis. In particular embodiments, the neurodegenerativedisease is Alzheimer's disease. In particular embodiments, theneurodegenerative disease is MS, such as primary progressive,relapsing-remitting secondary progressive or progressive relapsing MS.The subject may be, for example, a primate. The subject may be a human.

In particular embodiments of methods of treating or preventing aneurodegenerative disease in a subject, comprising administering to thesubject a pharmaceutically effective amount of a compound of the presentdisclosure, the treatment suppresses the demyelination of neurons in thesubject's brain or spinal cord. In certain embodiments, the treatmentsuppresses inflammatory demyelination. In certain embodiments, thetreatment suppresses the transection of neuron axons in the subject'sbrain or spinal cord. In certain embodiments, the treatment suppressesthe transection of neurites in the subject's brain or spinal cord. Incertain embodiments, the treatment suppresses neuronal apoptosis in thesubject's brain or spinal cord. In certain embodiments, the treatmentstimulates the remyelination of neuron axons in the subject's brain orspinal cord. In certain embodiments, the treatment restores lostfunction after an MS attack. In certain embodiments, the treatmentprevents a new MS attack. In certain embodiments, the treatment preventsa disability resulting from an MS attack.

One general aspect of the present disclosure contemplates a method oftreating or preventing a disorder characterized by overexpression ofiNOS genes in a subject, comprising administering to the subject apharmaceutically effective amount of a compound of the presentdisclosure.

Another general aspect of the present disclosure contemplates a methodof inhibiting IFN-γ-induced nitric oxide production in cells of asubject, comprising administering to said subject a pharmaceuticallyeffective amount of a compound of the present disclosure.

Yet another general method of the present disclosure contemplates amethod of treating or preventing a disorder characterized byoverexpression of COX-2 genes in a subject, comprising administering tothe subject a pharmaceutically effective amount of compound of thepresent disclosure.

Methods of treating renal/kidney disease (RKD) in a subject, comprisingadministering to the subject a pharmaceutically effective amount of acompound of the present disclosure are also contemplated. See U.S.patent application Ser. No. 12/352,473, which is incorporated byreference herein in its entirety. The RKD may result from, for example,a toxic insult. The toxic insult may result from, for example, animaging agent or a drug. The drug may be a chemotherapeutic, forexample. The RKD may result from ischemia/reperfusion injury, in certainembodiments. In certain embodiments, the RKD results from diabetes orhypertension. The RKD may result from an autoimmune disease. The RKD maybe further defined as chronic RKD, or acute RKD.

In certain methods of treating renal/kidney disease (RKD) in a subject,comprising administering to the subject a pharmaceutically effectiveamount of a compound of the present disclosure, the subject hasundergone or is undergoing dialysis. In certain embodiments, the subjecthas undergone or is a candidate to undergo kidney transplant. Thesubject may be a primate. The primate may be a human. The subject inthis or any other method may be, for example, a cow, horse, dog, cat,pig, mouse, rat or guinea pig.

Also contemplated by the present disclosure is a method for improvingglomerular filtration rate or creatinine clearance in a subject,comprising administering to the subject a pharmaceutically effectiveamount of a compound of the present disclosure.

In some embodiments, the invention provides compounds useful forpreventing and/or treating diseases or disorders whose pathologyinvolves oxidative stress, inflammation, and/or dysregulation ofinflammatory signaling pathways. In some variations, the diseases ordisorders can be characterized by overexpression of inducible nitricoxide synthase (iNOS) and/or inducible cyclooxygenase (COX-2) inaffected tissues. In some variations, the diseases or disorders can becharacterized by overproduction of reactive oxygen species (ROS) orreactive nitrogen species (RNS) such as superoxide, hydrogen peroxide,nitric oxide or peroxynitrite in affected tissues. In some variations,the disease or disorder is characterized by excessive production ofinflammatory cytokines or other inflammation-related proteins such asTNFα, IL-6, IL-1, IL-8, ICAM-1, VCAM-1, and VEGF. Such diseases ordisorders may, in some embodiments, involve undesirable proliferation ofcertain cells, as in the case of cancer (e.g., solid tumors, leukemias,myelomas, lymphomas, and other cancers), fibrosis associated with organfailure, or excessive scarring. Non limiting examples of the disease ordisorder include: lupus, rheumatoid arthritis, juvenile-onset diabetes,multiple sclerosis, psoriasis, and Crohn's disease. Further non-limitingexamples include cardiovascular diseases, such as atherosclerosis, heartfailure, myocardial infarction, acute coronary syndrome, restenosisfollowing vascular surgery, hypertension, and vasculitis;neurodegenerative or neuromuscular diseases such as Alzheimer's disease,Parkinson's disease, Huntington's disease, ALS, and muscular dystrophy;neurological disorders such as epilepsy and dystonia; neuropsychiatricconditions such as major depression, bipolar disorder, post-traumaticstress disorder, schizophrenia, anorexia nervosa, ADHD, andautism-spectrum disorders; retinal diseases such as maculardegeneration, diabetic retinopathy, glaucoma, and retinitis; chronic andacute pain syndromes, including inflammatory and neuropathic pain;hearing loss and tinnitus; diabetes and complications of diabetes,including metabolic syndrome, diabetic nephropathy, diabetic neuropathyand diabetic ulcers; respiratory diseases such as asthma, chronicobstructive pulmonary disease, acute respiratory distress syndrome, andcystic fibrosis; inflammatory bowel diseases; osteoporosis,osteoarthritis, and other degenerative conditions of bone and cartilage;acute or chronic organ failure, including renal failure, liver failure(including cirrhosis and hepatitis), and pancreatitis;ischemia-reperfusion injury associated with thrombotic or hemorrhagicstroke, subarachnoid hemorrhage, cerebral vasospasm, myocardialinfarction, shock, or trauma; complications of organ or tissuetransplantation including acute or chronic transplant failure orrejection and graft-versus-host disease; skin diseases including atopicdermatitis and acne; sepsis and septic shock; excessive inflammationassociated with infection, including respiratory inflammation associatedwith influenza and upper respiratory infections; mucositis associatedwith cancer therapy, including radiation therapy or chemotherapy; andsevere burns.

Methods of synthesizing compounds of the present disclosure are alsocontemplated. In particular embodiments, such methods can comprise amethod of making a target compound defined of the formula:

wherein R_(a) is alkoxy_((C1-4)), comprising reacting a compound of theformula:

with an oxidizing agent under a set of conditions to form the targetcompound.

Kits are also contemplated by the present disclosure, such as a kitcomprising: a compound of the present disclosure; and instructions whichcomprise one or more forms of information selected from the groupconsisting of indicating a disease state for which the compound is to beadministered, storage information for the compound, dosing informationand instructions regarding how to administer the compound. The kit maycomprise a compound of the present disclosure in a multiple dose form.

Other objects, features and advantages of the present disclosure willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.Note that simply because a particular compound is ascribed to oneparticular generic formula doesn't mean that it cannot also belong toanother generic formula.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentdisclosure. The invention may be better understood by reference to oneof these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIGS. 1-8 and 32-34. Inhibition of NO Production. RAW264.7 macrophageswere pre-treated with DMSO or drugs at various concentrations (nM) for 2hours, then treated with 20 ng/ml IFNγ for 24 hours. NO concentration inmedia was determined using a Griess reagent system; cell viability wasdetermined using WST-1 reagent.

FIG. 9. Suppression of COX-2 Induction. RAW264.7 cells were pre-treatedfor 2 hours with indicated compounds and subsequently stimulated with 10ng/ml IFNγ for an additional 24 hours. COX-2 protein levels were assayedby immunoblotting. Actin was used as a loading control. RTA 402 and RTA402 refer to comparison compounds 402 and 402 (see Example 1).

FIGS. 10-12. Inhibition of IL-6 Induced STAT3 Phosphorylation. HeLacells were treated with the indicated compounds and concentrations for 6hours and subsequently stimulated with 20 ng/ml IL-6 for 15 minutes.Phosphorylated STAT3 and total STAT3 levels were assayed byimmunoblotting. Compounds 402-52 and 402-53 are comparison compounds(see Example 1).

FIG. 13. Suppression of IL-6 Induced STAT3 Phosphorylation. HeLa cellswere treated with DMSO or the indicated compounds at 2 μM for 6 hoursand subsequently stimulated with 20 ng/ml IL-6 for 15 minutes.Phosphorylated STAT3 and total STAT3 levels were assayed byimmunoblotting. Compounds 402-54, 402-55 and 402-56 are comparisoncompounds (see Example 1).

FIG. 14. Inhibition of TNFα-induced IκBα degradation. HeLa cells weretreated with indicated compounds and concentrations for 6 hours andsubsequently stimulated with 20 ng/ml TNFα for 15 minutes. Lysates wereanalyzed with antibodies against IκBα and actin.

FIGS. 15 and 16. Inhibition of NFκB activation. HeLa cells weretransfected with pNF-κB-Luc (inducible) and pRL-TK (constitutive)reporter plasmids. Twenty-four hours later cells were pre-treated withthe indicated compounds for 2 hours. DMSO served as a vehicle control.Following pre-treatment, cells were stimulated with 20 ng/ml TNFα for 3hours. Reporter activity was measured using DualGlo luciferase reporterassay and pNF-κB luciferase activity was normalized against pRL-TKluciferase activity. Fold-induction of mean luciferase activity relativeto unstimulated (-TNFα) samples is shown. Error bars represent the SD ofthe mean of 6 samples.

FIGS. 17-20. Induction of HO-1. MDA-MB-435 human melanoma cells weretreated with vehicle (DMSO) or the indicated compounds andconcentrations for 16 hours. HO-1 mRNA levels were quantified using qPCRand were normalized relative to a DMSO-treated sample run in parallel.Values are averages of duplicate wells.

FIG. 21. Induction of HO-1, TrxR1 and γ-GCS. MDA-MB-435 human melanomacells were treated with vehicle (DMSO) or the indicated compounds andconcentrations for 16 hours. HO-1, thioredoxin reductase-1 (TrxR1), andγ-glutamylcysteine synthetase (γ-GCS) mRNA levels were quantified usingqPCR and were normalized relative to a DMSO-treated sample run inparallel. Values are averages of duplicate wells.

FIG. 22. Induction of TrxR1. MDA-MB-435 human melanoma cells weretreated with vehicle (DMSO) or the indicated compounds andconcentrations for 16 hours. Thioredoxin reductase-1 (TrxR1) mRNA levelswere quantified using qPCR and were normalized relative to aDMSO-treated sample run in parallel. Values are averages of duplicatewells. Compounds 401, 402-19 and 402-53 are comparison compounds (seeExample 1). Comparison with the results of FIG. 25 demonstrates thathigher concentrations of 402-02 and 404-02 are required to approacheffects seen with the unsaturated counterpart compounds 402 and 404.

FIG. 23. Induction of γ-GCS. MDA-MB-435 human melanoma cells weretreated with vehicle (DMSO) or the indicated compounds andconcentrations for 16 hours. γ-glutamylcysteine synthetase (γ-GCS) mRNAlevels were quantified using qPCR and were normalized relative to aDMSO-treated sample run in parallel. Values are averages of duplicatewells. Comparison with the results of FIG. 26 demonstrates that higherconcentrations of 402-02 and 404-02 are required to approach effectsseen with the unsaturated counterpart compounds 402 and 404.

FIG. 24. Induction of Ferritin Heavy Chain. MDA-MB-435 human melanomacells were treated with vehicle (DMSO) or the indicated compounds andconcentrations for 16 hours. Ferritin heavy chain mRNA levels werequantified using qPCR and were normalized relative to a DMSO-treatedsample run in parallel. Values are averages of duplicate wells.Comparison with the results of FIG. 27 demonstrates that higherconcentrations of 402-02 and 404-02 are required to approach effectsseen with the unsaturated counterpart compounds 402 and 404.

FIG. 25. Induction of TrxR1. MDA-MB-435 human melanoma cells weretreated with vehicle (DMSO) or the indicated compounds andconcentrations for 16 hours. Thioredoxin reductase-1 (TrxR1) mRNA levelswere quantified using qPCR and were normalized relative to aDMSO-treated sample run in parallel. Values are averages of duplicatewells. Comparison with the results of FIG. 22 demonstrates that higherconcentrations of 402-02 and 404-02 are required to approach effectsseen with the unsaturated counterpart compounds 402 and 404.

FIG. 26. Induction of γ-GCS. MDA-MB-435 human melanoma cells weretreated with vehicle (DMSO) or the indicated compounds andconcentrations for 16 hours. γ-glutamylcysteine synthetase (γ-GCS) mRNAlevels were quantified using qPCR and were normalized relative to aDMSO-treated sample run in parallel. Values are averages of duplicatewells. Comparison with the results of FIG. 23 demonstrates that higherconcentrations of 402-02 and 404-02 are required to approach effectsseen with the unsaturated counterpart compounds 402 and 404.

FIG. 27. Induction of Ferritin Heavy Chain. MDA-MB-435 human melanomacells were treated with vehicle (DMSO) or the indicated compounds andconcentrations for 16 hours. Ferritin heavy chain mRNA levels werequantified using qPCR and were normalized relative to a DMSO-treatedsample run in parallel. Values are averages of duplicate wells.Comparison with the results of FIG. 24 demonstrates that higherconcentrations of 402-02 and 404-02 are required to approach effectsseen with the unsaturated counterpart compounds 402 and 404.

FIG. 28—CDDO-TFEA (TP-500) Is Detected at Higher Levels in Mouse Brainthan CDDO-EA (TP-319). CD-1 mice were fed either 200 or 400 mg/kg dietof either TP-319 or TP-500 for 3.5 days, and TP levels in the brains ofthe mice were analyzed by LC/MS. The structures of TP-319 and TP-500 areshown below.

FIGS. 29 A & B—Weight Change Data from a Head to Head Tox Study ofCompounds 401 (FIG. 29A) versus 401-02 (FIG. 29B). Compounds wereassessed for toxicity in mice in a 14-day study. Each compound wasformulated in sesame oil and administered daily by oral gavage at dosesof 10, 50, 100, or 250 mg/kg (n=4 per group).

FIGS. 30 A & B—Weight Change Data from a Head to Head Tox Study ofCompounds 402 (FIG. 30A) versus 402-02 (FIG. 30B). Compounds wereassessed for toxicity in mice in a 14-day study. Each compound wasformulated in sesame oil and administered daily by oral gavage at dosesof 10, 50, 100, or 250 mg/kg (n=4 per group).

FIGS. 31A & B—Weight Change Data from a Head to Head Tox Study ofCompounds 402 (FIG. 31A) versus 404-02 (FIG. 31B). Compounds wereassessed for toxicity in mice in a 14-day study. Each compound wasformulated in sesame oil and administered daily by oral gavage at dosesof 10, 50, 100, or 250 mg/kg (n=4 per group).

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Disclosed herein are, for example, new compounds with antioxidant andanti-inflammatory properties, methods for their manufacture, and methodsfor their use, including for the treatment and/or prevention of disease.

I. DEFINITIONS

As used herein, “hydrogen” means —H; “hydroxy” means —OH; “oxo” means═O; “halo” means independently —F, —Cl, —Br or —I; “amino” means —NH₂(see below for definitions of groups containing the term amino, e.g.,alkylamino); “hydroxyamino” means —NHOH; “nitro” means —NO₂; imino means═NH (see below for definitions of groups containing the term imino,e.g., alkylamino); “cyano” means —CN; “azido” means —N₃; “mercapto”means —SH; “thio” means ═S; “sulfonamido” means —NHS(O)₂— (see below fordefinitions of groups containing the term sulfonamido, e.g.,alkylsulfonamido); “sulfonyl” means —S(O)₂— (see below for definitionsof groups containing the term sulfonyl, e.g., alkylsulfonyl); and“silyl” means —SiH₃ (see below for definitions of group(s) containingthe term silyl, e.g., alkylsilyl).

For the groups below, the following parenthetical subscripts furtherdefine the groups as follows: “(Cn)” defines the exact number (n) ofcarbon atoms in the group. “(C≦n)” defines the maximum number (n) ofcarbon atoms that can be in the group, with the minimum number of carbonatoms in such at least one, but otherwise as small as possible for thegroup in question. E.g., it is understood that the minimum number ofcarbon atoms in the group “alkenyl_((C≦8))” is 2. For example,“alkoxy_((C≦10))” designates those alkoxy groups having from 1 to 10carbon atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or any rangederivable therein (e.g., 3-10 carbon atoms)). (Cn−n′) defines both theminimum (n) and maximum number (n′) of carbon atoms in the group.Similarly, “alkyl_((C2-10))” designates those alkyl groups having from 2to 10 carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, or any rangederivable therein (e.g., 3-10 carbon atoms)).

The term “alkyl” when used without the “substituted” modifier refers toa non-aromatic monovalent group with a saturated carbon atom as thepoint of attachment, a linear or branched, cyclo, cyclic or acyclicstructure, no carbon-carbon double or triple bonds, and no atoms otherthan carbon and hydrogen. The groups, —CH₃ (Me), —CH₂CH₃ (Et),—CH₂CH₂CH₃ (n-Pr), —CH(CH₃)₂ (iso-Pr), —CH(CH₂)₂ (cyclopropyl),—CH₂CH₂CH₂CH₃ (n-Bu), —CH(CH₃)CH₂CH₃ (sec-butyl), —CH₂CH(CH₃)₂(iso-butyl), —C(CH₃)₃ (tert-butyl), —CH₂C(CH₃)₃ (neo-pentyl),cyclobutyl, cyclopentyl, cyclohexyl, and cyclohexylmethyl arenon-limiting examples of alkyl groups. The term “substituted alkyl”refers to a non-aromatic monovalent group with a saturated carbon atomas the point of attachment, a linear or branched, cyclo, cyclic oracyclic structure, no carbon-carbon double or triple bonds, and at leastone atom independently selected from the group consisting of N, O, F,Cl, Br, I, Si, P, and S. The following groups are non-limiting examplesof substituted alkyl groups: —CH₂OH, —CH₂Cl, —CH₂Br, —CH₂SH, —CF₃,—CH₂CN, —CH₂C(O)H, —CH₂C(O)OH, —CH₂C(O)OCH₃, —CH₂C(O)NH₂, —CH₂C(O)NHCH₃,—CH₂C(O)CH₃, —CH₂OCH₃, —CH₂OCH₂CF₃, —CH₂OC(O)CH₃, —CH₂NH₂, —CH₂NHCH₃,—CH₂N(CH₃)₂, —CH₂CH₂Cl, —CH₂CH₂OH, —CH₂CF₃, —CH₂CH₂OC(O)CH₃,—CH₂CH₂NHCO₂C(CH₃)₃, and —CH₂Si(CH₃)₃.

The term “alkanediyl” when used without the “substituted” modifierrefers to a non-aromatic divalent group, wherein the alkanediyl group isattached with two σ-bonds, with one or two saturated carbon atom(s) asthe point(s) of attachment, a linear or branched, cyclo, cyclic oracyclic structure, no carbon-carbon double or triple bonds, and no atomsother than carbon and hydrogen. The groups, —CH₂— (methylene), —CH₂CH₂—,—CH₂C(CH₃)₂CH₂, CH₂CH₂CH₂—, and

are non-limiting examples of alkanediyl groups. The term “substitutedalkanediyl” refers to a non-aromatic monovalent group, wherein thealkynediyl group is attached with two σ-bonds, with one or two saturatedcarbon atom(s) as the point(s) of attachment, a linear or branched,cyclo, cyclic or acyclic structure, no carbon-carbon double or triplebonds, and at least one atom independently selected from the groupconsisting of N, O, F, Cl, Br, I, Si, P, and S. The following groups arenon-limiting examples of substituted alkanediyl groups: —CH(F)—, —CF₂—,—CH(Cl)—, —CH(OH)—, —CH(OCH₃)—, and —CH₂CH(Cl)—.

The term “alkenyl” when used without the “substituted” modifier refersto a monovalent group with a nonaromatic carbon atom as the point ofattachment, a linear or branched, cyclo, cyclic or acyclic structure, atleast one nonaromatic carbon-carbon double bond, no carbon-carbon triplebonds, and no atoms other than carbon and hydrogen. Non-limitingexamples of alkenyl groups include: —CH═CH₂ (vinyl), —CH═CHCH₃,—CH═CHCH₂CH₃, —CH₂CH═CH₂ (allyl), —CH₂CH═CHCH₃, and —CH═CH—C₆H₅. Theterm “substituted alkenyl” refers to a monovalent group with anonaromatic carbon atom as the point of attachment, at least onenonaromatic carbon-carbon double bond, no carbon-carbon triple bonds, alinear or branched, cyclo, cyclic or acyclic structure, and at least oneatom independently selected from the group consisting of N, O, F, Cl,Br, I, Si, P, and S. The groups, —CH═CHF, —CH═CHCl and —CH═CHBr, arenon-limiting examples of substituted alkenyl groups.

The term “alkenediyl” when used without the “substituted” modifierrefers to a non-aromatic divalent group, wherein the alkenediyl group isattached with two σ-bonds, with two carbon atoms as points ofattachment, a linear or branched, cyclo, cyclic or acyclic structure, atleast one nonaromatic carbon-carbon double bond, no carbon-carbon triplebonds, and no atoms other than carbon and hydrogen. The groups, —CH═CH—,—CH═C(CH₃)CH₂—, —CH═CHCH₂—, and

are non-limiting examples of alkenediyl groups. The term “substitutedalkenediyl” refers to a non-aromatic divalent group, wherein thealkenediyl group is attached with two σ-bonds, with two carbon atoms aspoints of attachment, a linear or branched, cyclo, cyclic or acyclicstructure, at least one nonaromatic carbon-carbon double bond, nocarbon-carbon triple bonds, and at least one atom independently selectedfrom the group consisting of N, O, F, Cl, Br, I, Si, P, and S. Thefollowing groups are non-limiting examples of substituted alkenediylgroups: —CF═CH—, —C(OH)═CH—, and —CH₂CH═C(Cl)—.

The term “alkynyl” when used without the “substituted” modifier refersto a monovalent group with a nonaromatic carbon atom as the point ofattachment, a linear or branched, cyclo, cyclic or acyclic structure, atleast one carbon-carbon triple bond, and no atoms other than carbon andhydrogen. The groups, —C≡CH, —C≡CCH₃, —C≡CC₆H, and —CH₂C≡CCH₃, arenon-limiting examples of alkynyl groups. The term “substituted alkynyl”refers to a monovalent group with a nonaromatic carbon atom as the pointof attachment and at least one carbon-carbon triple bond, a linear orbranched, cyclo, cyclic or acyclic structure, and at least one atomindependently selected from the group consisting of N, O, F, Cl, Br, I,Si, P, and S. The group, —C≡CSi(CH₃)₃, is a non-limiting example of asubstituted alkynyl group.

The term “alkynediyl” when used without the “substituted” modifierrefers to a non-aromatic divalent group, wherein the alkynediyl group isattached with two σ-bonds, with two carbon atoms as points ofattachment, a linear or branched, cyclo, cyclic or acyclic structure, atleast one carbon-carbon triple bond, and no atoms other than carbon andhydrogen. The groups, —C≡C—, —C≡CCH₂—, and —C≡CCH(CH₃)— are non-limitingexamples of alkynediyl groups. The term “substituted alkynediyl” refersto a non-aromatic divalent group, wherein the alkynediyl group isattached with two σ-bonds, with two carbon atoms as points ofattachment, a linear or branched, cyclo, cyclic or acyclic structure, atleast one carbon-carbon triple bond, and at least one atom independentlyselected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S.The groups —C≡CCFH— and —C≡CHCH(Cl)— are non-limiting examples ofsubstituted alkynediyl groups.

The term “aryl” when used without the “substituted” modifier refers to amonovalent group with an aromatic carbon atom as the point ofattachment, said carbon atom forming part of a six-membered aromaticring structure wherein the ring atoms are all carbon, and wherein themonovalent group consists of no atoms other than carbon and hydrogen.Non-limiting examples of aryl groups include phenyl (Ph), methylphenyl,(dimethyl)phenyl, —C₆H₄CH₂CH₃ (ethylphenyl), —C₆H₄CH₂CH₂CH₃(propylphenyl), —C₆H₄CH(CH₃)₂, —C₆H₄CH(CH₂)₂, —C₆H₃(CH₃)CH₂CH₃(methylethylphenyl), —C₆H₄CH═CH₂ (vinylphenyl), —C₆H₄CH═CHCH₃,—C₆H₄C≡CH, —C₆H₄C≡CCH₃, naphthyl, and the monovalent group derived frombiphenyl. The term “substituted aryl” refers to a monovalent group withan aromatic carbon atom as the point of attachment, said carbon atomforming part of a six-membered aromatic ring structure wherein the ringatoms are all carbon, and wherein the monovalent group further has atleast one atom independently selected from the group consisting of N, O,F, Cl, Br, I, Si, P, and S, Non-limiting examples of substituted arylgroups include the groups: —C₆H₄F, —C₆H₄Cl, —C₆H₄Br, —C₆H₄I, —C₆H₄OH,—C₆H₄OCH₃, —C₆H₄OCH₂CH₃, —C₆H₄OC(O)CH₃, —C₆H₄NH₂, —C₆H₄NHCH₃,—C₆H₄N(CH₃)₂, —C₆H₄CH₂OH, —C₆H₄CH₂OC(O)CH₃, —C₆H₄CH₂NH₂, —C₆H₄CF₃,—C₆H₄CN, —C₆H₄CHO, C₆H₄CHO, C₆H₄C(O)CH₃, —C₆H₄C(O)C₆H₅, —C₆H₄CO₂H,—C₆H₄CO₂CH₃, —C₆H₄CONH₂, —C₆H₄CONHCH₃, and —C₆H₄CON(CH₃)₂.

The term “arenediyl” when used without the “substituted” modifier refersto a divalent group, wherein the arenediyl group is attached with twoσ-bonds, with two aromatic carbon atoms as points of attachment, saidcarbon atoms forming part of one or more six-membered aromatic ringstructure(s) wherein the ring atoms are all carbon, and wherein themonovalent group consists of no atoms other than carbon and hydrogen.Non-limiting examples of arenediyl groups include:

The term “substituted arenediyl” refers to a divalent group, wherein thearenediyl group is attached with two σ-bonds, with two aromatic carbonatoms as points of attachment, said carbon atoms forming part of one ormore six-membered aromatic rings structure(s), wherein the ring atomsare all carbon, and wherein the divalent group further has at least oneatom independently selected from the group consisting of N, O, F, Cl,Br, I, Si, P, and S.

The term “aralkyl” when used without the “substituted” modifier refersto the monovalent group-alkanediyl-aryl, in which the terms alkanediyland aryl are each used in a manner consistent with the definitionsprovided above. Non-limiting examples of aralkyls are: phenylmethyl(benzyl, Bn), 1-phenyl-ethyl, 2-phenyl-ethyl, indenyl and2,3-dihydro-indenyl, provided that indenyl and 2,3-dihydro-indenyl areonly examples of aralkyl in so far as the point of attachment in eachcase is one of the saturated carbon atoms. When the term “aralkyl” isused with the “substituted” modifier, either one or both the alkanediyland the aryl is substituted. Non-limiting examples of substitutedaralkyls are: (3-chlorophenyl)-methyl, 2-oxo-2-phenyl-ethyl(phenylcarbonylmethyl), 2-chloro-2-phenyl-ethyl, chromanyl where thepoint of attachment is one of the saturated carbon atoms, andtetrahydroquinolinyl where the point of attachment is one of thesaturated atoms.

The term “heteroaryl” when used without the “substituted” modifierrefers to a monovalent group with an aromatic carbon atom or nitrogenatom as the point of attachment, said carbon atom or nitrogen atomforming part of an aromatic ring structure wherein at least one of thering atoms is nitrogen, oxygen or sulfur, and wherein the monovalentgroup consists of no atoms other than carbon, hydrogen, aromaticnitrogen, aromatic oxygen and aromatic sulfur. Non-limiting examples ofaryl groups include acridinyl, furanyl, imidazoimidazolyl,imidazopyrazolyl, imidazopyridinyl, imidazopyrimidinyl, indolyl,indazolinyl, methylpyridyl, oxazolyl, phenylimidazolyl, pyridyl,pyrrolyl, pyrimidyl, pyrazinyl, quinolyl, quinazolyl, quinoxalinyl,tetrahydroquinolinyl, thienyl, triazinyl, pyrrolopyridinyl,pyrrolopyrimidinyl, pyrrolopyrazinyl, pyrrolotriazinyl,pyrroloimidazolyl, chromenyl (where the point of attachment is one ofthe aromatic atoms), and chromanyl (where the point of attachment is oneof the aromatic atoms). The term “substituted heteroaryl” refers to amonovalent group with an aromatic carbon atom or nitrogen atom as thepoint of attachment, said carbon atom or nitrogen atom forming part ofan aromatic ring structure wherein at least one of the ring atoms isnitrogen, oxygen or sulfur, and wherein the monovalent group further hasat least one atom independently selected from the group consisting ofnon-aromatic nitrogen, non-aromatic oxygen, non aromatic sulfur F, Cl,Br, I, Si, and P.

The term “heteroarenediyl” when used without the “substituted” modifierrefers to a divalent group, wherein the heteroarenediyl group isattached with two σ-bonds, with an aromatic carbon atom or nitrogen atomas the point of attachment, said carbon atom or nitrogen atom twoaromatic atoms as points of attachment, said carbon atoms forming partof one or more six-membered aromatic ring structure(s) wherein the ringatoms are all carbon, and wherein the monovalent group consists of noatoms other than carbon and hydrogen. Non-limiting examples ofheteroarenediyl groups include:

The term “substituted heteroarenediyl” refers to a divalent group,wherein the heteroarenediyl group is attached with two σ-bonds, with twoaromatic carbon atoms as points of attachment, said carbon atoms formingpart of one or more six-membered aromatic rings structure(s), whereinthe ring atoms are all carbon, and wherein the divalent group furtherhas at least one atom independently selected from the group consistingof N, O, F, Cl, Br, I, Si, P, and S.

The term “heteroaralkyl” when used without the “substituted” modifierrefers to the monovalent group-alkanediyl-heteroaryl, in which the termsalkanediyl and heteroaryl are each used in a manner consistent with thedefinitions provided above. Non-limiting examples of aralkyls are:pyridylmethyl, and thienylmethyl. When the term “heteroaralkyl” is usedwith the “substituted” modifier, either one or both the alkanediyl andthe heteroaryl is substituted.

The term “acyl” when used without the “substituted” modifier refers to amonovalent group with a carbon atom of a carbonyl group as the point ofattachment, further having a linear or branched, cyclo, cyclic oracyclic structure, further having no additional atoms that are notcarbon or hydrogen, beyond the oxygen atom of the carbonyl group. Thegroups, —CHO, —C(O)CH₃ (acetyl, Ac), —C(O)CH₂CH₃, —C(O)CH₂CH₂CH₃,—C(O)CH(CH₃)₂, —C(O)CH(CH₂)₂, —C(O)C₆H₅, —C(O)C₆H₄CH₃, —C(O)C₆H₄CH₂CH₃,—COC₆H₃(CH₃)₂, and —C(O)CH₂C₆H₅, are non-limiting examples of acylgroups. The term “acyl” therefore encompasses, but is not limited togroups sometimes referred to as “alkyl carbonyl” and “aryl carbonyl”groups. The term “substituted acyl” refers to a monovalent group with acarbon atom of a carbonyl group as the point of attachment, furtherhaving a linear or branched, cyclo, cyclic or acyclic structure, furtherhaving at least one atom, in addition to the oxygen of the carbonylgroup, independently selected from the group consisting of N, O, F, Cl,Br, I, Si, P, and S. The groups, —C(O)CH₂CF₃, —CO₂H (carboxyl), —CO₂CH₃(methylcarboxyl), —CO₂CH₂CH₃, —CO₂CH₂CH₂CH₃, CO₂C₆H₅, —CO₂CH(CH₃)₂,—CO₂CH(CH₂)₂, —C(O)NH₂ (carbamoyl), —C(O)NHCH₃, —C(O)NHCH₂CH₃,—CONHCH(CH₃)₂, —CONHCH(CH₂)₂, —CON(CH₃)₂, —CONHCH₂CF₃, —CO-pyridyl,—CO-imidazoyl, and —C(O)N₃, are non-limiting examples of substitutedacyl groups. The term “substituted acyl” encompasses, but is not limitedto, “heteroaryl carbonyl” groups.

The term “alkylidene” when used without the “substituted” modifierrefers to the divalent group ═CRR′, wherein the alkylidene group isattached with one σ-bond and one π-bond, in which R and R′ areindependently hydrogen, alkyl, or R and R′ are taken together torepresent alkanediyl. Non-limiting examples of alkylidene groupsinclude: ═CH₂, ═CH(CH₂CH₃), and ═C(CH₃)₂. The term “substitutedalkylidene” refers to the group ═CRR′, wherein the alkylidene group isattached with one σ-bond and one π-bond, in which R and R′ areindependently hydrogen, alkyl, substituted alkyl, or R and R′ are takentogether to represent a substituted alkanediyl, provided that either oneof R and R′ is a substituted alkyl or R and R′ are taken together torepresent a substituted alkanediyl.

The term “alkoxy” when used without the “substituted” modifier refers tothe group —OR, in which R is an alkyl, as that term is defined above.Non-limiting examples of alkoxy groups include: —OCH₃, —OCH₂CH₃,—OCH₂CH₂CH₃, —OCH(CH₃)₂, —OCH(CH₂)₂, —O-cyclopentyl, and —O-cyclohexyl.The term “substituted alkoxy” refers to the group —OR, in which R is asubstituted alkyl, as that term is defined above. For example, —OCH₂CF₃is a substituted alkoxy group.

Similarly, the terms “alkenyloxy”, “alkynyloxy”, “aryloxy”, “aralkoxy”,“heteroaryloxy”, “heteroaralkoxy” and “acyloxy”, when used without the“substituted” modifier, refers to groups, defined as —OR, in which R isalkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl and acyl,respectively, as those terms are defined above. When any of the termsalkenyloxy, alkynyloxy, aryloxy, aralkyloxy and acyloxy is modified by“substituted,” it refers to the group —OR, in which R is substitutedalkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl and acyl,respectively.

The term “alkylamino” when used without the “substituted” modifierrefers to the group —NHR, in which R is an alkyl, as that term isdefined above. Non-limiting examples of alkylamino groups include:—NHCH₃, —NHCH₂CH₃, —NHCH₂CH₂CH₃, —NHCH(CH₃)₂, —NHCH(CH₂)₂,—NHCH₂CH₂CH₂CH₃, —NHCH(CH₃)CH₂CH₃, —NHCH₂CH(CH₃)₂, —NHC(CH₃)₃,—NH-cyclopentyl, and —NH-cyclohexyl. The term “substituted alkylamino”refers to the group —NHR, in which R is a substituted alkyl, as thatterm is defined above. For example, —NHCH₂CF₃ is a substitutedalkylamino group.

The term “dialkylamino” when used without the “substituted” modifierrefers to the group —NRR′, in which R and R′ can be the same ordifferent alkyl groups, or R and R′ can be taken together to representan alkanediyl having two or more saturated carbon atoms, at least two ofwhich are attached to the nitrogen atom. Non-limiting examples ofdialkylamino groups include: —NHC(CH₃)₃, —N(CH₃)CH₂CH₃, —N(CH₂CH₃)₂,N-pyrrolidinyl, and N-piperidinyl. The term “substituted dialkylamino”refers to the group —NRR′, in which R and R′ can be the same ordifferent substituted alkyl groups, one of R or R′ is an alkyl and theother is a substituted alkyl, or R and R′ can be taken together torepresent a substituted alkanediyl with two or more saturated carbonatoms, at least two of which are attached to the nitrogen atom.

The terms “alkoxyamino”, “alkenylamino”, “alkynylamino”, “arylamino”,“aralkylamino”, “heteroarylamino”, “heteroaralkylamino”, and“alkylsulfonylamino” when used without the “substituted” modifier,refers to groups, defined as —NHR, in which R is alkoxy, alkenyl,alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl and alkylsulfonyl,respectively, as those terms are defined above. A non-limiting exampleof an arylamino group is —NHC₆H₅. When any of the terms alkoxyamino,alkenylamino, alkynylamino, arylamino, aralkylamino, heteroarylamino,hetero aralkylamino and alkylsulfonylamino is modified by “substituted,”it refers to the group —NHR, in which R is substituted alkoxy, alkenyl,alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl and alkylsulfonyl,respectively.

The term “amido” (acylamino), when used without the “substituted”modifier, refers to the group —NHR, in which R is acyl, as that term isdefined above. A non-limiting example of an acylamino group is—NHC(O)CH₃. When the term amido is used with the “substituted” modifier,it refers to groups, defined as —NHR, in which R is substituted acyl, asthat term is defined above. The groups —NHC(O)OCH₃ and —NHC(O)NHCH₃ arenon-limiting examples of substituted amido groups.

The term “alkylimino” when used without the “substituted” modifierrefers to the group ═NR, wherein the alkylimino group is attached withone σ-bond and one π-bond, in which R is an alkyl, as that term isdefined above. Non-limiting examples of alkylimino groups include:═NCH₃, ═NCH₂CH₃ and ═N-cyclohexyl. The term “substituted alkylimino”refers to the group ═NR, wherein the alkylimino group is attached withone σ-bond and one π-bond, in which R is a substituted alkyl, as thatterm is defined above. For example, ═NCH₂CF₃ is a substituted alkyliminogroup.

Similarly, the terms “alkenylimino”, “alkynylimino”, “arylimino”,“aralkylimino”, “heteroarylimino”, “heteroaralkylimino” and “acylimino”,when used without the “substituted” modifier, refers to groups, definedas ═NR, wherein the alkylimino group is attached with one σ-bond and oneπ-bond, in which R is alkenyl, alkynyl, aryl, aralkyl, heteroaryl,heteroaralkyl and acyl, respectively, as those terms are defined above.When any of the terms alkenylimino, alkynylimino, arylimino,aralkylimino and acylimino is modified by “substituted,” it refers tothe group ═NR, wherein the alkylimino group is attached with one σ-bondand one π-bond, in which R is substituted alkenyl, alkynyl, aryl,aralkyl, heteroaryl, heteroaralkyl and acyl, respectively.

The term “fluoroalkyl” when used without the “substituted” modifierrefers to an alkyl, as that term is defined above, in which one or morefluorines have been substituted for hydrogens. The groups, —CH₂F, —CF₃,and —CH₂CF₃ are non-limiting examples of fluoroalkyl groups. The term“substituted fluoroalkyl” refers to a non-aromatic monovalent group witha saturated carbon atom as the point of attachment, a linear orbranched, cyclo, cyclic or acyclic structure, at least one fluorineatom, no carbon-carbon double or triple bonds, and at least one atomindependently selected from the group consisting of N, O, Cl, Br, I, Si,P, and S. The following group is a non-limiting example of a substitutedfluoroalkyl: —CFHOH.

The term “alkylthio” when used without the “substituted” modifier refersto the group —SR, in which R is an alkyl, as that term is defined above.Non-limiting examples of alkylthio groups include: —SCH₃, —SCH₂CH₃,—SCH₂CH₂CH₃, —SCH(CH₃)₂, —SCH(CH₂)₂, —S-cyclopentyl, and —S-cyclohexyl.The term “substituted alkylthio” refers to the group —SR, in which R isa substituted alkyl, as that term is defined above. For example,—SCH₂CF₃ is a substituted alkylthio group.

Similarly, the terms “alkenylthio”, “alkynylthio”, “arylthio”,“aralkylthio”, “heteroarylthio”, “heteroaralkylthio”, and “acylthio”,when used without the “substituted” modifier, refers to groups, definedas —SR, in which R is alkenyl, alkynyl, aryl, aralkyl, heteroaryl,heteroaralkyl and acyl, respectively, as those terms are defined above.When any of the terms alkenylthio, alkynylthio, arylthio, aralkylthio,heteroarylthio, heteroaralkylthio, and acylthio is modified by“substituted,” it refers to the group —SR, in which R is substitutedalkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl and acyl,respectively.

The term “thioacyl” when used without the “substituted” modifier refersto a monovalent group with a carbon atom of a thiocarbonyl group as thepoint of attachment, further having a linear or branched, cyclo, cyclicor acyclic structure, further having no additional atoms that are notcarbon or hydrogen, beyond the sulfur atom of the carbonyl group. Thegroups, —CHS, —C(S)CH₃, —C(S)CH₂CH₃, —C(S)CH₂CH₂CH₃, —C(S)CH(CH₃)₂,—C(S)CH(CH₂)₂, —C(S)C₆H₅, —C(S)C₆H₄CH₃, —C(S)C₆H₄CH₂CH₃,—C(S)C₆H₃(CH₃)₂, and —C(S)CH₂C₆H₅, are non-limiting examples of thioacylgroups. The term “thioacyl” therefore encompasses, but is not limitedto, groups sometimes referred to as “alkyl thiocarbonyl” and “arylthiocarbonyl” groups. The term “substituted thioacyl” refers to aradical with a carbon atom as the point of attachment, the carbon atombeing part of a thiocarbonyl group, further having a linear or branched,cyclo, cyclic or acyclic structure, further having at least one atom, inaddition to the sulfur atom of the carbonyl group, independentlyselected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S.The groups, —C(S)CH₂CF₃, —C(S)O₂H, —C(S)OCH₃, —C(S)OCH₂CH₃,—C(S)OCH₂CH₂CH₃, —C(S)OC₆H₅, —C(S)OCH(CH₃)₂, —C(S)OCH(CH₂)₂, —C(S)NH₂,and —C(S)NHCH₃, are non-limiting examples of substituted thioacylgroups. The term “substituted thioacyl” encompasses, but is not limitedto, “heteroaryl thiocarbonyl” groups.

The term “alkylsulfonyl” when used without the “substituted” modifierrefers to the group —S(O)₂R, in which R is an alkyl, as that term isdefined above. Non-limiting examples of alkylsulfonyl groups include:—S(O)₂CH₃, —S(O)₂CH₂CH₃, —S(O)₂CH₂CH₂CH₃, —S(O)₂CH(CH₃)₂,—S(O)₂CH(CH₂)₂, —S(O)₂-cyclopentyl, and —S(O)₂-cyclohexyl. The term“substituted alkylsulfonyl” refers to the group —S(O)₂R, in which R is asubstituted alkyl, as that term is defined above. For example,—S(O)₂CH₂CF₃ is a substituted alkylsulfonyl group.

Similarly, the terms “alkenylsulfonyl”, “alkynylsulfonyl”,“arylsulfonyl”, “aralkylsulfonyl”, “heteroarylsulfonyl”, and“heteroaralkylsulfonyl” when used without the “substituted” modifier,refers to groups, defined as —S(O)₂R, in which R is alkenyl, alkynyl,aryl, aralkyl, heteroaryl, and heteroaralkyl, respectively, as thoseterms are defined above. When any of the terms alkenylsulfonyl,alkynylsulfonyl, arylsulfonyl, aralkylsulfonyl, heteroarylsulfonyl, andheteroaralkylsulfonyl is modified by “substituted,” it refers to thegroup —S(O)₂R, in which R is substituted alkenyl, alkynyl, aryl,aralkyl, heteroaryl and heteroaralkyl, respectively.

The term “alkylammonium” when used without the “substituted” modifierrefers to a group, defined as —NH₂R⁺, —NHRR′⁺, or —NRR′R″⁺, in which R,R′ and R″ are the same or different alkyl groups, or any combination oftwo of R, R′ and R″ can be taken together to represent an alkanediyl.Non-limiting examples of alkylammonium cation groups include:—NH₂(CH₃)⁺, —NH₂(CH₂CH₃)+, —NH₂(CH₂CH₂CH₃)+, —NH(CH₃)₂ ⁺, —NH(CH₂CH₃)₂⁺, —NH(CH₂CH₂CH₃)₂ ⁺, —N(CH₃)₃ ⁺, —N(CH₃)(CH₂CH₃)₂ ⁺, —N(CH₃)₂(CH₂CH₃)⁺,—NH₂C(CH₃)₃ ⁺, —NH(cyclopentyl)₂ ⁺, and —NH₂(cyclohexyl)⁺. The term“substituted alkylammonium” refers —NH₂R⁺, —NHRR′⁺, or —NRR′R″⁺, inwhich at least one of R, R′ and R″ is a substituted alkyl or two of R,R′ and R″ can be taken together to represent a substituted alkanediyl.When more than one of R, R′ and R″ is a substituted alkyl, they can bethe same of different. Any of R, R′ and R″ that are not eithersubstituted alkyl or substituted alkanediyl, can be either alkyl, eitherthe same or different, or can be taken together to represent aalkanediyl with two or more carbon atoms, at least two of which areattached to the nitrogen atom shown in the formula.

The term “alkylsulfonium” when used without the “substituted” modifierrefers to the group —SRR′⁺, in which R and R′ can be the same ordifferent alkyl groups, or R and R′ can be taken together to representan alkanediyl. Non-limiting examples of alkylsulfonium groups include:—SH(CH₃)⁺, —SH(CH₂CH₃)⁺, —SH(CH₂CH₂CH₃)⁺, —S(CH₃)₂ ⁺, —S(CH₂CH₃)₂ ⁺,—S(CH₂CH₂CH₃)₂ ⁺, —SH(cyclopentyl)⁺, and —SH(cyclohexyl)⁺. The term“substituted alkylsulfonium” refers to the group —SRR′⁺, in which R andR′ can be the same or different substituted alkyl groups, one of R or R′is an alkyl and the other is a substituted alkyl, or R and R′ can betaken together to represent a substituted alkanediyl. For example,—SH(CH₂CF₃)⁺ is a substituted alkylsulfonium group.

The term “alkylsilyl” when used without the “substituted” modifierrefers to a monovalent group, defined as —SiH₂R, —SiHRR′, or —SiRR′R″,in which R, R′ and R″ can be the same or different alkyl groups, or anycombination of two of R, R′ and R″ can be taken together to represent analkanediyl. The groups, —SiH₂CH₃, —SiH(CH₃)₂, —Si(CH₃)₃ and—Si(CH₃)₂C(CH₃)₃, are non-limiting examples of unsubstituted alkylsilylgroups. The term “substituted alkylsilyl” refers —SiH₂R, —SiHRR′, or—SiRR′R″, in which at least one of R, R′ and R″ is a substituted alkylor two of R, R′ and R″ can be taken together to represent a substitutedalkanediyl. When more than one of R, R′ and R″ is a substituted alkyl,they can be the same of different. Any of R, R′ and R″ that are noteither substituted alkyl or substituted alkanediyl, can be either alkyl,either the same or different, or can be taken together to represent aalkanediyl with two or more saturated carbon atoms, at least two ofwhich are attached to the silicon atom.

In addition, atoms making up the compounds of the present disclosure areintended to include all isotopic forms of such atoms. Isotopes, as usedherein, include those atoms having the same atomic number but differentmass numbers. By way of general example and without limitation, isotopesof hydrogen include tritium and deuterium, and isotopes of carboninclude ¹³C and ¹⁴C. Similarly, it is contemplated that one or morecarbon atom(s) of a compound of the present disclosure may be replacedby a silicon atom(s). Furthermore, it is contemplated that one or moreoxygen atom(s) of a compound of the present disclosure may be replacedby a sulfur or selenium atom(s).

A compound having a formula that is represented with a dashed bond isintended to include the formulae optionally having zero, one or moredouble bonds. Thus, for example, the structure

includes the structures

As will be understood by a person of skill in the art, no one such ringatom forms part of more than one double bond.

Any undefined valency on an atom of a structure shown in thisapplication implicitly represents a hydrogen atom bonded to the atom.

A ring structure shown with an unconnected “R” group, indicates that anyimplicit hydrogen atom on that ring can be replaced with that R group.In the case of a divalent R group (e.g., oxo, imino, thio, alkylidene,etc.), any pair of implicit hydrogen atoms attached to one atom of thatring can be replaced by that R group. This concept is as exemplifiedbelow:

represents

As used herein, a “chiral auxiliary” refers to a removable chiral groupthat is capable of influencing the stereoselectivity of a reaction.Persons of skill in the art are familiar with such compounds, and manyare commercially available.

The use of the word “a” or “an,” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects.

The terms “comprise,” “have” and “include” are open-ended linking verbs.Any forms or tenses of one or more of these verbs, such as “comprises,”“comprising,” “has,” “having,” “includes” and “including,” are alsoopen-ended. For example, any method that “comprises,” “has” or“includes” one or more steps is not limited to possessing only those oneor more steps and also covers other unlisted steps.

The term “effective,” as that term is used in the specification and/orclaims, means adequate to accomplish a desired, expected, or intendedresult.

The term “hydrate” when used as a modifier to a compound means that thecompound has less than one (e.g., hemihydrate), one (e.g., monohydrate),or more than one (e.g., dihydrate) water molecules associated with eachcompound molecule, such as in solid forms of the compound.

As used herein, the term “IC₅₀” refers to an inhibitory dose which is50% of the maximum response obtained.

An “isomer” of a first compound is a separate compound in which eachmolecule contains the same constituent atoms as the first compound, butwhere the configuration of those atoms in three dimensions differs.

As used herein, the term “patient” or “subject” refers to a livingmammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat,mouse, rat, guinea pig, or transgenic species thereof. In certainembodiments, the patient or subject is a primate. Non-limiting examplesof human subjects are adults, juveniles, infants and fetuses.

“Pharmaceutically acceptable” means that which is useful in preparing apharmaceutical composition that is generally safe, non-toxic and neitherbiologically nor otherwise undesirable and includes that which isacceptable for veterinary use as well as human pharmaceutical use.

“Pharmaceutically acceptable salts” means salts of compounds of thepresent disclosure which are pharmaceutically acceptable, as definedabove, and which possess the desired pharmacological activity. Suchsalts include acid addition salts formed with inorganic acids such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like; or with organic acids such as1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid,2-naphthalenesulfonic acid, 3-phenylpropionic acid,4,4′-methylenebis(3-hydroxy-2-ene-1-carboxylic acid),4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, acetic acid,aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acids,aromatic sulfuric acids, benzenesulfonic acid, benzoic acid,camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid,cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid,glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid,heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid,laurylsulfuric acid, maleic acid, malic acid, malonic acid, mandelicacid, methanesulfonic acid, muconic acid, o-(4-hydroxybenzoyl)benzoicacid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substitutedalkanoic acids, propionic acid, p-toluenesulfonic acid, pyruvic acid,salicylic acid, stearic acid, succinic acid, tartaric acid,tertiarybutylacetic acid, trimethylacetic acid, and the like.Pharmaceutically acceptable salts also include base addition salts whichmay be formed when acidic protons present are capable of reacting withinorganic or organic bases. Acceptable inorganic bases include sodiumhydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide andcalcium hydroxide. Acceptable organic bases include ethanolamine,diethanolamine, triethanolamine, tromethamine, N-methylglucamine and thelike. It should be recognized that the particular anion or cationforming a part of any salt of this invention is not critical, so long asthe salt, as a whole, is pharmacologically acceptable. Additionalexamples of pharmaceutically acceptable salts and their methods ofpreparation and use are presented in Handbook of Pharmaceutical Salts:Properties, and Use (2002).

As used herein, “predominantly one enantiomer” means that a compoundcontains at least about 85% of one enantiomer, or more preferably atleast about 90% of one enantiomer, or even more preferably at leastabout 95% of one enantiomer, or most preferably at least about 99% ofone enantiomer. Similarly, the phrase “substantially free from otheroptical isomers” means that the composition contains at most about 15%of another enantiomer or diastereomer, more preferably at most about 10%of another enantiomer or diastereomer, even more preferably at mostabout 5% of another enantiomer or diastereomer, and most preferably atmost about 1% of another enantiomer or diastereomer.

“Prevention” or “preventing” includes: (1) inhibiting the onset of adisease in a subject or patient which may be at risk and/or predisposedto the disease but does not yet experience or display any or all of thepathology or symptomatology of the disease, and/or (2) slowing the onsetof the pathology or symptomatology of a disease in a subject or patientwhich may be at risk and/or predisposed to the disease but does not yetexperience or display any or all of the pathology or symptomatology ofthe disease.

“Prodrug” means a compound that is convertible in vivo metabolicallyinto an inhibitor according to the present disclosure. The prodrugitself may or may not also have activity with respect to a given targetprotein. For example, a compound comprising a hydroxy group may beadministered as an ester that is converted by hydrolysis in vivo to thehydroxy compound. Suitable esters that may be converted in vivo intohydroxy compounds include acetates, citrates, lactates, phosphates,tartrates, malonates, oxalates, salicylates, propionates, succinates,fumarates, maleates, methylene-bis-β-hydroxynaphthoate, gentisates,isethionates, di-p-toluoyltartrates, methane-sulfonates,ethanesulfonates, benzenesulfonates, p-toluenesulfonates,cyclohexyl-sulfamates, quinates, esters of amino acids, and the like.Similarly, a compound comprising an amine group may be administered asan amide that is converted by hydrolysis in vivo to the amine compound.

The term “saturated” when referring to an atom means that the atom isconnected to other atoms only by means of single bonds.

A “stereoisomer” or “optical isomer” is an isomer of a given compound inwhich the same atoms are bonded to the same other atoms, but where theconfiguration of those atoms in three dimensions differs. “Enantiomers”are stereoisomers of a given compound that are mirror images of eachother, like left and right hands. “Diastereomers” are stereoisomers of agiven compound that are not enantiomers.

The invention contemplates that for any stereocenter or axis ofchirality for which stereochemistry has not been defined, thatstereocenter or axis of chirality can be present in its R form, S form,or as a mixture of the R and S forms, including racemic and non-racemicmixtures.

“Substituent convertible to hydrogen in vivo” means any group that isconvertible to a hydrogen atom by enzymological or chemical meansincluding, but not limited to, hydrolysis and hydrogenolysis. Examplesinclude acyl groups, groups having an oxycarbonyl group, amino acidresidues, peptide residues, o-nitrophenylsulfenyl, trimethylsilyl,tetrahydro-pyranyl, diphenylphosphinyl, hydroxy or alkoxy substituentson imino groups, and the like. Examples of acyl groups include formyl,acetyl, trifluoroacetyl, and the like. Examples of groups having anoxycarbonyl group include ethoxycarbonyl, tert-butoxycarbonyl(—C(O)OC(CH₃)₃), benzyloxycarbonyl, p-methoxy-benzyloxycarbonyl,vinyloxycarbonyl, β-(p-toluenesulfonyl)ethoxycarbonyl, and the like.Suitable amino acid residues include, but are not limited to, residuesof Gly (glycine), Ala (alanine), Arg (arginine), Asn (asparagine), Asp(aspartic acid), Cys (cysteine), Glu (glutamic acid), His (histidine),Ile (isoleucine), Leu (leucine), Lys (lysine), Met (methionine), Phe(phenylalanine), Pro (proline), Ser (serine), Thr (threonine), Trp(tryptophan), Tyr (tyrosine), Val (valine), Nva (norvaline), Hse(homoserine), 4-Hyp (4-hydroxyproline), 5-Hyl (5-hydroxylysine), Orn(ornithine) and β-Ala. Examples of suitable amino acid residues alsoinclude amino acid residues that are protected with a protecting group.Examples of suitable protecting groups include those typically employedin peptide synthesis, including acyl groups (such as formyl and acetyl),arylmethyloxycarbonyl groups (such as benzyloxycarbonyl andp-nitrobenzyloxycarbonyl), tert-butoxycarbonyl groups (—C(O)OC(CH₃)₃),and the like. Suitable peptide residues include peptide residuescomprising two to five, and optionally amino acid residues. The residuesof these amino acids or peptides can be present in stereochemicalconfigurations of the D-form, the L-form or mixtures thereof. Inaddition, the amino acid or peptide residue may have an asymmetriccarbon atom. Examples of suitable amino acid residues having anasymmetric carbon atom include residues of Ala, Leu, Phe, Trp, Nva, Val,Met, Ser, Lys, Thr and Tyr. Peptide residues having an asymmetric carbonatom include peptide residues having one or more constituent amino acidresidues having an asymmetric carbon atom. Examples of suitable aminoacid protecting groups include those typically employed in peptidesynthesis, including acyl groups (such as formyl and acetyl),arylmethyloxycarbonyl groups (such as benzyloxycarbonyl andp-nitrobenzyloxycarbonyl), tert-butoxycarbonyl groups (—C(O)OC(CH₃)₃),and the like. Other examples of substituents “convertible to hydrogen invivo” include reductively eliminable hydrogenolyzable groups. Examplesof suitable reductively eliminable hydrogenolyzable groups include, butare not limited to, arylsulfonyl groups (such as o-toluenesulfonyl);methyl groups substituted with phenyl or benzyloxy (such as benzyl,trityl and benzyloxymethyl); arylmethoxycarbonyl groups (such asbenzyloxycarbonyl and o-methoxy-benzyloxycarbonyl); andhaloethoxycarbonyl groups (such as β,β,β-trichloroethoxycarbonyl andβ-iodoethoxycarbonyl).

“Therapeutically effective amount” or “pharmaceutically effectiveamount” means that amount which, when administered to a subject orpatient for treating a disease, is sufficient to effect such treatmentfor the disease.

“Treatment” or “treating” includes (1) inhibiting a disease in a subjector patient experiencing or displaying the pathology or symptomatology ofthe disease (e.g., arresting further development of the pathology and/orsymptomatology), (2) ameliorating a disease in a subject or patient thatis experiencing or displaying the pathology or symptomatology of thedisease (e.g., reversing the pathology and/or symptomatology), and/or(3) effecting any measurable decrease in a disease in a subject orpatient that is experiencing or displaying the pathology orsymptomatology of the disease.

As used herein, the term “water soluble” means that the compounddissolves in water at least to the extent of 0.010 mole/liter or isclassified as soluble according to literature precedence.

Other abbreviations used herein are as follows: DMSO, dimethylsulfoxide; NO, nitric oxide; iNOS, inducible nitric oxide synthase;COX-2, cyclooxygenase-2; NGF, nerve growth factor; IBMX,isobutylmethylxanthine; FBS, fetal bovine serum; GPDH, glycerol3-phosphate dehydrogenase; RXR, retinoid X receptor; TGF-β, transforminggrowth factor-β; IFNγ or IFN-γ, interferon-γ; LPS, bacterial endotoxiclipopolysaccharide; TNFα or TNF-α, tumor necrosis factor-α; IL-1β,interleukin-1β; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MTT,3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; TCA,trichloroacetic acid; HO-1, inducible heme oxygenase.

The above definitions supersede any conflicting definition in any of thereference that is incorporated by reference herein. The fact thatcertain terms are defined, however, should not be considered asindicative that any term that is undefined is indefinite. Rather, allterms used are believed to describe the invention in terms such that oneof ordinary skill can appreciate the scope and practice the presentdisclosure.

II. SYNTHETIC METHODS

Compounds of the present disclosure may be made using the methodsoutlined in the Examples section (Example 2 and 3). These methods can befurther modified and optimized using the principles and techniques oforganic chemistry as applied by a person skilled in the art. Suchprinciples and techniques are taught, for example, in March's AdvancedOrganic Chemistry: Reactions, Mechanisms, and Structure (2007), which isincorporated by reference herein.

III. BIOLOGICAL ACTIVITY OF OLEANOLIC ACID DERIVATIVES

Biological activity results, both in vivo and in vitro are providedthroughout the present disclosure. These include: inhibition of NOProduction, suppression of COX-2 induction, inhibition of IL-6 inducedSTAT3 phosphorylation, suppression of IL-6 induced STAT3phosphorylation, inhibition of TNFα-induced IκBα degradation, inhibitionof NFκB activation, induction of HO-1, Nrf2 induction of HO-1, TrxR1 andγ-GCS, induction of TrxR1, induction of γ-GCS, induction of ferritinheavy chain, induction of TrxR1, induction of γ-GCS, induction offerritin heavy chain, and various in vivo toxicity studies. See figuresand figure descriptions. Suppression of NO production and induction ofNrf2 induction results can be respectively summarized as shown Tables 1aand 1b, below. Further results, including toxicity studies, are providedin the Examples section.

TABLE 1a Suppression of IFNγ-Induced NO Production. RAW264.7 (20 ng/mlIFNγ) iNOS suppr. Compound ID(s) MW NO IC₅₀ WST-1 IC₅₀ WB63101/402-02/dh402 507.70 ~12 nM  200 nM >90% 63102/404-02/dh404 574.72~45 nM >200 nM 63250/402-46 509.70 >200 nM >200 nM 63197/402-48512.72 >200 nM >200 nM 63195/402-49 509.72 >200 nM >200 nM63196/402-51/dh401 493.68 ~75 nM >200 nM 63252/402-57 474.68 ~5 nM >200nM 63205/402-59 492.69 ~25 nM >200 nM 63206/402-64 477.68 ~10 nM >200 nM63207/402-66 509.72 ~50 nM >200 nM 63219/402-78 509.72 ~150 nM >200 nM63229 517.71 >200 nM >200 nM 63230 531.74 ~50 nM >200 nM 63227576.73 >200 nM >200 nM 63219 509.72 ~150 nM >200 nM 63223 576.73 >200nM >200 nM 63237 572.70 ~80 nM >200 nM 63268 576.75 >200 nM >200 nM63274 547.81 ~70 nM >200 nM 63289 525.73 >200 nM >200 nM 63295 522.73~20 nM >200 nM 63296 522.73 ~200 nM >200 nM 63308 491.70 ~25 nM >200 nM63323 583.80 ~40 nM See FIG. 33 63325 520.75 ~50 nM See FIG. 32 63326552.79 ~50 nM See FIG. 34

TABLE 1b Induction of HO-1, TrxR1 and γ-GCS in Human Melanoma Cells.Nrf2 target gene induction in MDA-MB-435 cells Compound 400 nM* 250 nM**Code HO-1 TrxR1 γ-GCS HO-1 NQO1 γ-GCS 63101 4 47 53 3 2 5 (dh402) 631023 2 4.5 (dh404) 63196 1 48 26 (dh401) 63252 5 3 63205 1.7 1.7 3.5 632062.5 1.8 4.5 63207 1.2 1.6 3.1 63237 3 2.3 6.4 Blank entry: Notdetermined. *Data expressed as a percent of induction observed for 402(see below for structure). **Data expressed as fold induction above DMSOcontrol.

In certain embodiments, the compounds of the present disclosure arecapable of crossing the blood brain barrier and achievingtherapeutically effective concentrations in the brain. They maytherefore be used to treat neurodegenerative diseases, brain cancer andother inflammatory conditions affecting the central nervous system. Forexample, 404-02 has been shown to cross the blood-brain barrier andachieve high concentrations in the central nervous system tissuefollowing oral dosing. Like the other compounds of the presentdisclosure, it promotes the resolution of innate and adaptiveimmune-mediated inflammation by restoring redox homeostasis in inflamedtissues. It is a potent inducer of the antioxidant transcription factorNrf2 and inhibitor of the pro-oxidant/pro-inflammatory transcriptionfactors NF-κB and the STATs. These biological pathways are implicated ina wide variety of diseases, including autoimmune conditions and severalneuro degenerative diseases.

IV. IMPROVED RODENT TOXICOLOGY

In certain embodiments, the invention provides compounds possessing lowtoxicity in rodents. In some cases, toxicity in rodents has beenobserved in preclinical studies with some analogues containing acarbon-carbon double bond in the C-ring, including 402 and 401.Compounds having a saturated C-ring, in contrast, have consistentlyshown low toxicity in rodents. Predictably low rodent toxicity providesan advantage since high rodent toxicity can be a significantcomplication in conducting preclinical studies required for developmentand registration of therapeutic compounds for use in humans or non-humananimals. Illustrations of this effect are provided below.

For example, an initial study (Example 6) was performed in SpragueDawley rats using both 402 and 402-02 and showed that 402-02 was lesstoxic. In a further study (Example 7), six compounds (401, 402, 404,401-2, 402-2, and 404-2) were assessed for toxicity in mice in a 14-daystudy. At higher doses (above 10 mg/kg/day) both 401 and 402 caused atleast 50% mortality, while 402 was non-toxic. In contrast, no mortalitywas observed in the 402-2 and 404-2 groups and only the highest dose of401-02 caused any lethality (Table 5). Body weight measurements (FIGS.29-31) were consistent with the mortality observations. Notably, the twohighest doses of 401 and 402 were lethal within 4 days, in contrast tothe effects of 401-2 and 402-2.

V. DISEASES ASSOCIATED WITH INFLAMMATION AND/OR OXIDATIVE STRESS

Inflammation is a biological process that provides resistance toinfectious or parasitic organisms and the repair of damaged tissue.Inflammation is commonly characterized by localized vasodilation,redness, swelling, and pain, the recruitment of leukocytes to the siteof infection or injury, production of inflammatory cytokines such asTNF-α and IL-1, and production of reactive oxygen or nitrogen speciessuch as hydrogen peroxide, superoxide and peroxynitrite. In later stagesof inflammation, tissue remodeling, angiogenesis, and scar formation(fibrosis) may occur as part of the wound healing process. Under normalcircumstances, the inflammatory response is regulated and temporary andis resolved in an orchestrated fashion once the infection or injury hasbeen dealt with adequately. However, acute inflammation can becomeexcessive and life-threatening if regulatory mechanisms fail.Alternatively, inflammation can become chronic and cause cumulativetissue damage or systemic complications.

Many serious and intractable human diseases involve dysregulation ofinflammatory processes, including diseases such as cancer,atherosclerosis, and diabetes, which were not traditionally viewed asinflammatory conditions. In the case of cancer, the inflammatoryprocesses are associated with tumor formation, progression, metastasis,and resistance to therapy. Atherosclerosis, long viewed as a disorder oflipid metabolism, is now understood to be primarily an inflammatorycondition, with activated macrophages playing an important role in theformation and eventual rupture of atherosclerotic plaques. Activation ofinflammatory signaling pathways has also been shown to play a role inthe development of insulin resistance, as well as in the peripheraltissue damage associated with diabetic hyperglycemia. Excessiveproduction of reactive oxygen species and reactive nitrogen species suchas superoxide, hydrogen peroxide, nitric oxide, and peroxynitrite is ahallmark of inflammatory conditions. Evidence of dysregulatedperoxynitrite production has been reported in a wide variety of diseases(Szabo et al., 2007; Schulz et al., 2008; Forstermann, 2006; Pall,2007).

Autoimmune diseases such as rheumatoid arthritis, lupus, psoriasis, andmultiple sclerosis involve inappropriate and chronic activation ofinflammatory processes in affected tissues, arising from dysfunction ofself vs. non-self recognition and response mechanisms in the immunesystem. In neurodegenerative diseases such as Alzheimer's andParkinson's diseases, neural damage is correlated with activation ofmicroglia and elevated levels of pro-inflammatory proteins such asinducible nitric oxide synthase (iNOS). Chronic organ failure such asrenal failure, heart failure, and chronic obstructive pulmonary diseaseis closely associated with the presence of chronic oxidative stress andinflammation, leading to the development of fibrosis and eventual lossof organ function.

Many other disorders involve oxidative stress and inflammation inaffected tissues, including inflammatory bowel disease; inflammatoryskin diseases; mucositis related to radiation therapy and chemotherapy;eye diseases such as uveitis, glaucoma, macular degeneration, andvarious forms of retinopathy; transplant failure and rejection;ischemia-reperfusion injury; chronic pain; degenerative conditions ofthe bones and joints including osteoarthritis and osteoporosis; asthmaand cystic fibrosis; seizure disorders; and neuropsychiatric conditionsincluding schizophrenia, depression, bipolar disorder, post-traumaticstress disorder, attention deficit disorders, autism-spectrum disorders,and eating disorders such as anorexia nervosa. Dysregulation ofinflammatory signaling pathways is believed to be a major factor in thepathology of muscle wasting diseases including muscular dystrophy andvarious forms of cachexia.

A variety of life-threatening acute disorders also involve dysregulatedinflammatory signaling, including acute organ failure involving thepancreas, kidneys, liver, or lungs, myocardial infarction or acutecoronary syndrome, stroke, septic shock, trauma, severe burns, andanaphylaxis.

Many complications of infectious diseases also involve dysregulation ofinflammatory responses. Although an inflammatory response can killinvading pathogens, an excessive inflammatory response can also be quitedestructive and in some cases can be a primary source of damage ininfected tissues. Furthermore, an excessive inflammatory response canalso lead to systemic complications due to overproduction ofinflammatory cytokines such as TNF-α and IL-1. This is believed to be afactor in mortality arising from severe influenza, severe acuterespiratory syndrome, and sepsis.

The aberrant or excessive expression of either iNOS or cyclooxygenase-2(COX-2) has been implicated in the pathogenesis of many diseaseprocesses. For example, it is clear that NO is a potent mutagen (Tamirand Tannebaum, 1996), and that nitric oxide can also activate COX-2(Salvemini et al., 1994). Furthermore, there is a marked increase iniNOS in rat colon tumors induced by the carcinogen, azoxymethane(Takahashi et al., 1997). A series of synthetic triterpenoid analogs ofoleanolic acid have been shown to be powerful inhibitors of cellularinflammatory processes, such as the induction by IFN-γ of induciblenitric oxide synthase (iNOS) and of COX-2 in mouse macrophages. SeeHonda et al. (2000a); Honda et al. (2000b), and Honda et al. (2002),which are all incorporated herein by reference.

In one aspect, compounds of the invention are characterized by theirability to inhibit the production of nitric oxide in macrophage-derivedRAW 264.7 cells induced by exposure to γ-interferon. They are furthercharacterized by their ability to induce the expression of antioxidantproteins such as NQO1 and reduce the expression of pro-inflammatoryproteins such as COX-2 and inducible nitric oxide synthase (iNOS). Theseproperties are relevant to the treatment of a wide array of diseasesinvolving oxidative stress and dysregulation of inflammatory processesincluding cancer, mucositis resulting from radiation therapy orchemotherapy, autoimmune diseases, cardiovascular diseases includingatherosclerosis, ischemia-reperfusion injury, acute and chronic organfailure including renal failure and heart failure, respiratory diseases,diabetes and complications of diabetes, severe allergies, transplantrejection, graft-versus-host disease, neurodegenerative diseases,diseases of the eye and retina, acute and chronic pain, degenerativebone diseases including osteoarthritis and osteoporosis, inflammatorybowel diseases, dermatitis and other skin diseases, sepsis, burns,seizure disorders, and neuropsychiatric disorders.

Without being bound by theory, the activation of theantioxidant/anti-inflammatory Keap1/Nrf2/ARE pathway is believed to beimplicated in both the anti-inflammatory and anti-carcinogenicproperties of the present oleanolic acid derivatives.

In another aspect, compounds of the invention may be used for treating asubject having a condition caused by elevated levels of oxidative stressin one or more tissues. Oxidative stress results from abnormally high orprolonged levels of reactive oxygen species such as superoxide, hydrogenperoxide, nitric oxide, and peroxynitrite (formed by the reaction ofnitric oxide and superoxide). The oxidative stress may be accompanied byeither acute or chronic inflammation. The oxidative stress may be causedby mitochondrial dysfunction, by activation of immune cells such asmacrophages and neutrophils, by acute exposure to an external agent suchas ionizing radiation or a cytotoxic chemotherapy agent (e.g.,doxorubicin), by trauma or other acute tissue injury, byischemia/reperfusion, by poor circulation or anemia, by localized orsystemic hypoxia or hyperoxia, by elevated levels of inflammatorycytokines and other inflammation-related proteins, and/or by otherabnormal physiological states such as hyperglycemia or hypoglycemia.

In animal models of many such conditions, stimulating expression ofinducible heme oxygenase (HO-1), a target gene of the Nrf2 pathway, hasbeen shown to have a significant therapeutic effect including models ofmyocardial infarction, renal failure, transplant failure and rejection,stroke, cardiovascular disease, and autoimmune disease (e.g., Sacerdotiet al., 2005; Abraham & Kappas, 2005; Bach, 2006; Araujo et al., 2003;Liu et al., 2006; Ishikawa et al., 2001; Kruger et al., 2006; Satoh etal., 2006; Zhou et al., 2005; Morse and Choi, 2005; Morse and Choi,2002). This enzyme breaks free heme down into iron, carbon monoxide(CO), and biliverdin (which is subsequently converted to the potentantioxidant molecule, bilirubin).

In another aspect, compounds of this invention may be used in preventingor treating tissue damage or organ failure, acute and chronic, resultingfrom oxidative stress exacerbated by inflammation. Examples of diseasesthat fall in this category include: heart failure, liver failure,transplant failure and rejection, renal failure, pancreatitis, fibroticlung diseases (cystic fibrosis and COPD, among others), diabetes(including complications), atherosclerosis, ischemia-reperfusion injury,glaucoma, stroke, autoimmune disease, autism, macular degeneration, andmuscular dystrophy. For example, in the case of autism, studies suggestthat increased oxidative stress in the central nervous system maycontribute to the development of the disease (Chauhan and Chauhan,2006).

Evidence also links oxidative stress and inflammation to the developmentand pathology of many other disorders of the central nervous system,including psychiatric disorders such as psychosis, major depression, andbipolar disorder; seizure disorders such as epilepsy; pain and sensorysyndromes such as migraine, neuropathic pain or tinnitus; and behavioralsyndromes such as the attention deficit disorders. See, e.g., Dickersonet al., 2007; Hanson et al., 2005; Kendall-Tackett, 2007; Lencz et al.,2007; Dudhgaonkar et al., 2006; Lee et al., 2007; Morris et al., 2002;Ruster et al., 2005; McIver et al., 2005; Sarchielli et al., 2006;Kawakami et al., 2006; Ross et al., 2003, which are all incorporated byreference herein. For example, elevated levels of inflammatorycytokines, including TNF, interferon-γ, and IL-6, are associated withmajor mental illness (Dickerson et al., 2007). Microglial activation hasalso been linked to major mental illness. Therefore, downregulatinginflammatory cytokines and inhibiting excessive activation of microgliacould be beneficial in patients with schizophrenia, major depression,bipolar disorder, autism-spectrum disorders, and other neuropsychiatricdisorders.

Accordingly, in pathologies involving oxidative stress alone oroxidative stress exacerbated by inflammation, treatment may compriseadministering to a subject a therapeutically effective amount of acompound of this invention, such as those described above or throughoutthis specification. Treatment may be administered preventively, inadvance of a predictable state of oxidative stress (e.g., organtransplantation or the administration of radiation therapy to a cancerpatient), or it may be administered therapeutically in settingsinvolving established oxidative stress and inflammation.

The compounds of the invention may be generally applied to the treatmentof inflammatory conditions, such as sepsis, dermatitis, autoimmunedisease and osteoarthritis. In one aspect, the compounds of thisinvention may be used to treat inflammatory pain and/or neuropathicpain, for example, by inducing Nrf2 and/or inhibiting NF-κB.

In one aspect, the compounds of the invention may be used to function asantioxidant inflammation modulators (AIMs) having potentanti-inflammatory properties that mimic the biological activity ofcyclopentenone prostaglandins (cyPGs). In one embodiment, the compoundsof the invention may be used to control the production ofpro-inflammatory cytokines by selectively targeting regulatory cysteineresidues (RCRs) on proteins that regulate the transcriptional activityof redox-sensitive transcription factors. Activation of RCRs by cyPGs orAIMs has been shown to initiate a pro-resolution program in which theactivity of the antioxidant and cytoprotective transcription factor Nrf2is potently induced, and the activities of the pro-oxidant andpro-inflammatory transcription factors NF-κB and the STATs aresuppressed. This increases the production of antioxidant and reductivemolecules (e.g., NQO1, HO-1, SOD1, and/or γ-GCS) and/or decreasesoxidative stress and the production of pro-oxidant and pro-inflammatorymolecules (e.g., iNOS, COX-2, and/or TNF-α).

In some embodiments, the compounds of the invention may be used in thetreatment and prevention of diseases such as cancer, inflammation,Alzheimer's disease, Parkinson's disease, multiple sclerosis, autism,amyotrophic lateral sclerosis, autoimmune diseases such as rheumatoidarthritis, lupus, and MS, inflammatory bowel disease, all other diseaseswhose pathogenesis is believed to involve excessive production of eithernitric oxide or prostaglandins, and pathologies involving oxidativestress alone or oxidative stress exacerbated by inflammation.

Another aspect of inflammation is the production of inflammatoryprostaglandins such as prostaglandin E. These molecules promotevasodilation, plasma extravasation, localized pain, elevatedtemperature, and other symptoms of inflammation. The inducible form ofthe enzyme COX-2 is associated with their production, and high levels ofCOX-2 are found in inflamed tissues. Consequently, inhibition of COX-2may relieve many symptoms of inflammation and a number of importantanti-inflammatory drugs (e.g., ibuprofen and celecoxib) act byinhibiting COX-2 activity. Recent research, however, has demonstratedthat a class of cyclopentenone prostaglandins (cyPGs) (e.g., 15-deoxyprostaglandin J2, a.k.a. PGJ2) plays a role in stimulating theorchestrated resolution of inflammation (e.g., Rajakariar et al., 2007).COX-2 is also associated with the production of cyclopentenoneprostaglandins. Consequently, inhibition of COX-2 may interfere with thefull resolution of inflammation, potentially promoting the persistenceof activated immune cells in tissues and leading to chronic,“smoldering” inflammation. This effect may be responsible for theincreased incidence of cardiovascular disease in patients usingselective COX-2 inhibitors for long periods of time.

In one aspect, the compounds of the invention may be used to control theproduction of pro-inflammatory cytokines within the cell by selectivelyactivating regulatory cysteine residues (RCRs) on proteins that regulatethe activity of redox-sensitive transcription factors. Activation ofRCRs by cyPGs has been shown to initiate a pro-resolution program inwhich the activity of the antioxidant and cytoprotective transcriptionfactor Nrf2 is potently induced and the activities of the pro-oxidantand pro-inflammatory transcription factors NF-κB and the STATs aresuppressed. In some embodiments, this increases the production ofantioxidant and reductive molecules (NQO1, HO-1, SOD1, γ-GCS) anddecreases oxidative stress and the production of pro-oxidant andpro-inflammatory molecules (iNOS, COX-2, TNF-α). In some embodiments,the compounds of this invention may cause the cells that host theinflammatory event to revert to a non-inflammatory state by promotingthe resolution of inflammation and limiting excessive tissue damage tothe host.

A. Cancer

Further, the compounds of the present disclosure may be used to induceapoptosis in tumor cells, to induce cell differentiation, to inhibitcancer cell proliferation, to inhibit an inflammatory response, and/orto function in a chemopreventative capacity. For example, the inventionprovides new compounds that have one or more of the followingproperties: (1) an ability to induce apoptosis and differentiate bothmalignant and non-malignant cells, (2) an activity at sub-micromolar ornanomolar levels as an inhibitor of proliferation of many malignant orpremalignant cells, (3) an ability to suppress the de novo synthesis ofthe inflammatory enzyme inducible nitric oxide synthase (iNOS), (4) anability to inhibit NF-κB activation, and (5) an ability to induce theexpression of heme oxygenase-1 (HO-1).

The levels of iNOS and COX-2 are elevated in certain cancers and havebeen implicated in carcinogenesis and COX-2 inhibitors have been shownto reduce the incidence of primary colonic adenomas in humans (Rostom etal., 2007; Brown and DuBois, 2005; Crowel et al., 2003). iNOS isexpressed in myeloid-derived suppressor cells (MDSCs) (Angulo et al.,2000) and COX-2 activity in cancer cells has been shown to result in theproduction of prostaglandin E₂ (PGE₂), which has been shown to inducethe expression of arginase in MDSCs (Sinha et al., 2007). Arginase andiNOS are enzymes that utilize L-arginine as a substrate and produceL-ornithine and urea, and L-citrulline and NO, respectively. Thedepletion of arginine from the tumor microenvironment by MDSCs, combinedwith the production of NO and peroxynitrite has been shown to inhibitproliferation and induce apoptosis of T cells (Bronte et al., 2003)Inhibition of COX-2 and iNOS has been shown to reduce the accumulationof MDSCs, restore cytotoxic activity of tumor-associated T cells, anddelay tumor growth (Sinha et al., 2007; Mazzoni et al., 2002; Zhou etal., 2007).

Inhibition of the NF-κB and JAK/STAT signaling pathways has beenimplicated as a strategy to inhibit proliferation of cancer epithelialcells and induce their apoptosis. Activation of STAT3 and NF-κB has beenshown to result in suppression of apoptosis in cancer cells, andpromotion of proliferation, invasion, and metastasis. Many of the targetgenes involved in these processes have been shown to betranscriptionally regulated by both NF-κB and STAT3 (Yu et al., 2007).

In addition to their direct roles in cancer epithelial cells, NF-κB andSTAT3 also have important roles in other cells found within the tumormicroenvironment. Experiments in animal models have demonstrated thatNF-κB is required in both cancer cells and hematopoeitic cells topropagate the effects of inflammation on cancer initiation andprogression (Greten et al., 2004). NF-κB inhibition in cancer andmyeloid cells reduces the number and size, respectively, of theresultant tumors. Activation of STAT3 in cancer cells results in theproduction of several cytokines (IL-6, IL-10) which suppress thematuration of tumor-associated dendritic cells (DC). Furthermore, STAT3is activated by these cytokines in the dendritic cells themselvesInhibition of STAT3 in mouse models of cancer restores DC maturation,promotes antitumor immunity, and inhibits tumor growth (Kortylewski etal., 2005).

B. Treatment of Multiple Sclerosis and Other NeurodegenerativeConditions

The compounds and methods of this invention may be used for treatingpatients for multiple sclerosis (MS). MS is known to be an inflammatorycondition of the central nervous system (Williams et al., 1994; Merrilland Benvenist, 1996; Genain and Nauser, 1997). Based on severalinvestigations, there is evidence suggesting that inflammatory,oxidative, and/or immune mechanisms are involved in the pathogenesis ofAlzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateralsclerosis (ALS), and MS (Bagasra et al., 1995; McGeer and McGeer, 1995;Simonian and Coyle, 1996; Kaltschmidt et al., 1997). Both reactiveastrocytes and activated microglia have been implicated in causation ofneurodegenerative disease (NDD) and neuroinflammatory disease (NID);there has been a particular emphasis on microglia as cells thatsynthesize both NO and prostaglandins as products of the respectiveenzymes, iNOS and COX-2. De novo formation of these enzymes may bedriven by inflammatory cytokines such as interferon-γ or interleukin-1.In turn, excessive production of NO may lead to inflammatory cascadesand/or oxidative damage in cells and tissues of many organs, includingneurons and oligodendrocytes of the nervous system, with consequentmanifestations in AD and MS, and possible PD and ALS (Coyle andPuttfarcken, 1993; Beal, 1996; Merrill and Benvenist, 1996; Simonian andCoyle, 1996; Vodovotz et al., 1996). Epidemiologic data indicate thatchronic use of NSAID's which block synthesis of prostaglandins fromarachidonate, markedly lower the risk for development of AD (McGeer etal., 1996; Stewart et al., 1997). Thus, agents that block formation ofNO and prostaglandins, may be used in approaches to prevention andtreatment of NDD. Successful therapeutic candidates for treating such adisease typically require an ability to penetrate the blood-brainbarrier. See, for example, U.S. Patent Publication 2009/0060873, whichis incorporated by reference herein in its entirety. See also, forexample, the results presented for compound 404-02 in Examples 4 and 5,below.

C. Neuroinflammation

The compounds and methods of this invention may be used for treatingpatients with neuroinflammation. Neuroinflammation encapsulates the ideathat microglial and astrocytic responses and actions in the centralnervous system have a fundamentally inflammation-like character, andthat these responses are central to the pathogenesis and progression ofa wide variety of neurological disorders. This idea originated in thefield of Alzheimer's disease (Griffin et al., 1989; Rogers et al.,1988), where it has revolutionized our understanding of this disease(Akiyama et al., 2000). These ideas have been extended to otherneurodegenerative diseases (Eikelenboom et al., 2002; Ishizawa andDickson, 2001), to ischemic/toxic diseases (Gehrmann et al., 1995;Touzani et al., 1999), to tumor biology (Graeber et al., 2002) and evento normal brain development.

Neuroinflammation incorporates a wide spectrum of complex cellularresponses that include activation of microglia and astrocytes andinduction of cytokines, chemokines, complement proteins, acute phaseproteins, oxidative injury, and related molecular processes. Theseevents may have detrimental effects on neuronal function, leading toneuronal injury, further glial activation, and ultimatelyneurodegeneration.

D. Treatment of Renal Failure

The compounds and methods of this invention may be used for treatingpatients with renal failure. See U.S. patent application Ser. No.12/352,473, which is incorporated by reference herein in its entirety.Another aspect of the present disclosure concerns new methods andcompounds for the treatment and prevention of renal disease. Renalfailure, resulting in inadequate clearance of metabolic waste productsfrom the blood and abnormal concentrations of electrolytes in the blood,is a significant medical problem throughout the world, especially indeveloped countries. Diabetes and hypertension are among the mostimportant causes of chronic renal failure, also known as chronic kidneydisease (CKD), but it is also associated with other conditions such aslupus. Acute renal failure may arise from exposure to certain drugs(e.g., acetaminophen) or toxic chemicals, or from ischemia-reperfusioninjury associated with shock or surgical procedures such astransplantation, and may result in chronic renal failure. In manypatients, renal failure advances to a stage in which the patientrequires regular dialysis or kidney transplantation to continue living.Both of these procedures are highly invasive and associated withsignificant side effects and quality of life issues. Although there areeffective treatments for some complications of renal failure, such ashyperparathyroidism and hyperphosphatemia, no available treatment hasbeen shown to halt or reverse the underlying progression of renalfailure. Thus, agents that can improve compromised renal function wouldrepresent a significant advance in the treatment of renal failure.

Inflammation contributes significantly to the pathology of CKD. There isalso a strong mechanistic link between oxidative stress and renaldysfunction. The NF-κB signaling pathway plays an important role in theprogression of CKD as NF-κB regulates the transcription of MCP-1, achemokine that is responsible for the recruitment ofmonocytes/macrophages resulting in an inflammatory response thatultimately injures the kidney (Wardle, 2001). The Keap1/Nrf2/ARE pathwaycontrols the transcription of several genes encoding antioxidantenzymes, including heme oxygenase-1 (HO-1). Ablation of the Nrf2 gene infemale mice results in the development of lupus-like glomerularnephritis (Yoh et al., 2001). Furthermore, several studies havedemonstrated that HO-1 expression is induced in response to renal damageand inflammation and that this enzyme and its products—bilirubin andcarbon monoxide—play a protective role in the kidney (Nath et al.,2006).

The glomerulus and the surrounding Bowman's capsule constitute the basicfunctional unit of the kidney. Glomerular filtration rate (GFR) is thestandard measure of renal function. Creatinine clearance is commonlyused to measure GFR. However, the level of serum creatinine is commonlyused as a surrogate measure of creatinine clearance. For instance,excessive levels of serum creatinine are generally accepted to indicateinadequate renal function and reductions in serum creatinine over timeare accepted as an indication of improved renal function. Normal levelsof creatinine in the blood are approximately 0.6 to 1.2 milligrams (mg)per deciliter (dl) in adult males and 0.5 to 1.1 milligrams perdeciliter in adult females.

Acute kidney injury (AKI) can occur following ischemia-reperfusion,treatment with certain pharmacological agents such as cisplatin andrapamycin, and intravenous injection of radiocontrast media used inmedical imaging. As in CKD, inflammation and oxidative stress contributeto the pathology of AKI. The molecular mechanisms underlyingradiocontrast-induced nephropathy (RCN) are not well understood;however, it is likely that a combination of events including prolongedvasoconstriction, impaired kidney autoregulation, and direct toxicity ofthe contrast media all contribute to renal failure (Tumlin et al.,2006). Vasoconstriction results in decreased renal blood flow and causesischemia-reperfusion and the production of reactive oxygen species. HO-1is strongly induced under these conditions and has been demonstrated toprevent ischemia-reperfusion injury in several different organs,including the kidney (Nath et al., 2006). Specifically, induction ofHO-1 has been shown to be protective in a rat model of RCN (Goodman etal., 2007). Reperfusion also induces an inflammatory response, in partthough activation of NF-κB signaling (Nichols, 2004). Targeting NF-κBhas been proposed as a therapeutic strategy to prevent organ damage(Zingarelli et al., 2003).

E. Cardiovascular Disease

The compounds and methods of this invention may be used for treatingpatients with cardiovascular disease. See U.S. patent application Ser.No. 12/352,473, which is incorporated by reference herein in itsentirety. Cardiovascular (CV) disease is among the most important causesof mortality worldwide, and is the leading cause of death in manydeveloped nations. The etiology of CV disease is complex, but themajority of causes are related to inadequate or completely disruptedsupply of blood to a critical organ or tissue. Frequently such acondition arises from the rupture of one or more atheroscleroticplaques, which leads to the formation of a thrombus that blocks bloodflow in a critical vessel. Such thrombosis is the principal cause ofheart attacks, in which one or more of the coronary arteries is blockedand blood flow to the heart itself is disrupted. The resulting ischemiais highly damaging to cardiac tissue, both from lack of oxygen duringthe ischemic event and from excessive formation of free radicals afterblood flow is restored (a phenomenon known as ischemia-reperfusioninjury). Similar damage occurs in the brain during a thrombotic stroke,when a cerebral artery or other major vessel is blocked by thrombosis.Hemorrhagic strokes, in contrast, involve rupture of a blood vessel andbleeding into the surrounding brain tissue. This creates oxidativestress in the immediate area of the hemorrhage, due to the presence oflarge amounts of free heme and other reactive species, and ischemia inother parts of the brain due to compromised blood flow. Subarachnoidhemorrhage, which is frequently accompanied by cerebral vasospasm, alsocauses ischemia/reperfusion injury in the brain.

Alternatively, atherosclerosis may be so extensive in critical bloodvessels that stenosis (narrowing of the arteries) develops and bloodflow to critical organs (including the heart) is chronicallyinsufficient. Such chronic ischemia can lead to end-organ damage of manykinds, including the cardiac hypertrophy associated with congestiveheart failure.

Atherosclerosis, the underlying defect leading to many forms ofcardiovascular disease, occurs when a physical defect or injury to thelining (endothelium) of an artery triggers an inflammatory responseinvolving the proliferation of vascular smooth muscle cells and theinfiltration of leukocytes into the affected area. Ultimately, acomplicated lesion known as an atherosclerotic plaque may form, composedof the above-mentioned cells combined with deposits ofcholesterol-bearing lipoproteins and other materials (e.g., Hansson etal., 2006).

Pharmaceutical treatments for cardiovascular disease include preventivetreatments, such as the use of drugs intended to lower blood pressure orcirculating levels of cholesterol and lipoproteins, as well astreatments designed to reduce the adherent tendencies of platelets andother blood cells (thereby reducing the rate of plaque progression andthe risk of thrombus formation). More recently, drugs such asstreptokinase and tissue plasminogen activator have been introduced andare used to dissolve the thrombus and restore blood flow. Surgicaltreatments include coronary artery bypass grafting to create analternative blood supply, balloon angioplasty to compress plaque tissueand increase the diameter of the arterial lumen, and carotidendarterectomy to remove plaque tissue in the carotid artery. Suchtreatments, especially balloon angioplasty, may be accompanied by theuse of stents, expandable mesh tubes designed to support the arterywalls in the affected area and keep the vessel open. Recently, the useof drug-eluting stents has become common in order to preventpost-surgical restenosis (renarrowing of the artery) in the affectedarea. These devices are wire stents coated with a biocompatible polymermatrix containing a drug that inhibits cell proliferation (e.g.,paclitaxel or rapamycin). The polymer allows a slow, localized releaseof the drug in the affected area with minimal exposure of non-targettissues. Despite the significant benefits offered by such treatments,mortality from cardiovascular disease remains high and significant unmetneeds in the treatment of cardiovascular disease remain.

As noted above, induction of HO-1 has been shown to be beneficial in avariety of models of cardiovascular disease, and low levels of HO-1expression have been clinically correlated with elevated risk of CVdisease. Compounds of the invention, therefore, may be used in treatingor preventing a variety of cardiovascular disorders including but notlimited to atherosclerosis, hypertension, myocardial infarction, chronicheart failure, stroke, subarachnoid hemorrhage, and restenosis.

F. Diabetes

The compounds and methods of this invention may be used for treatingpatients with diabetes. See U.S. patent application Ser. No. 12/352,473,which is incorporated by reference herein in its entirety. Diabetes is acomplex disease characterized by the body's failure to regulatecirculating levels of glucose. This failure may result from a lack ofinsulin, a peptide hormone that regulates both the production andabsorption of glucose in various tissues. Deficient insulin compromisesthe ability of muscle, fat, and other tissues to absorb glucoseproperly, leading to hyperglycemia (abnormally high levels of glucose inthe blood). Most commonly, such insulin deficiency results frominadequate production in the islet cells of the pancreas. In themajority of cases this arises from autoimmune destruction of thesecells, a condition known as type 1 or juvenile-onset diabetes, but mayalso be due to physical trauma or some other cause.

Diabetes may also arise when muscle and fat cells become less responsiveto insulin and do not absorb glucose properly, resulting inhyperglycemia. This phenomenon is known as insulin resistance, and theresulting condition is known as Type 2 diabetes. Type 2 diabetes, themost common type, is highly associated with obesity and hypertension.Obesity is associated with an inflammatory state of adipose tissue thatis thought to play a major role in the development of insulin resistance(e.g., Hotamisligil, 2006; Guilherme et al., 2008).

Diabetes is associated with damage to many tissues, largely becausehyperglycemia (and hypoglycemia, which can result from excessive orpoorly timed doses of insulin) is a significant source of oxidativestress. Chronic kidney failure, retinopathy, peripheral neuropathy,peripheral vasculitis, and the development of dermal ulcers that healslowly or not at all are among the common complications of diabetes.Because of their ability to protect against oxidative stress,particularly by the induction of HO-1 expression, compounds of theinvention may be used in treatments for many complications of diabetes.As noted above (Cai et al., 2005), chronic inflammation and oxidativestress in the liver are suspected to be primary contributing factors inthe development of Type 2 diabetes. Furthermore, PPARγ agonists such asthiazolidinediones are capable of reducing insulin resistance and areknown to be effective treatments for Type 2 diabetes.

The effect of treatment of diabetes may be evaluated as follows. Boththe biological efficacy of the treatment modality as well as theclinical efficacy are evaluated, if possible. For example, because thedisease manifests itself by increased blood sugar, the biologicalefficacy of the treatment therefore can be evaluated, for example, byobservation of return of the evaluated blood glucose towards normal.Measurement of glycosylated hemoglobin, also called A1c or HbA1c, isanother commonly used parameter of blood glucose control. Measuring aclinical endpoint which can give an indication of b-cell regenerationafter, for example, a six-month period of time, can give an indicationof the clinical efficacy of the treatment regimen.

G. Rheumatoid Arthritis

The compounds and methods of this invention may be used for treatingpatients with RA. Typically the first signs of rheumatoid arthritis (RA)appear in the synovial lining layer, with proliferation of synovialfibroblasts and their attachment to the articular surface at the jointmargin (Lipsky, 1998). Subsequently, macrophages, T cells and otherinflammatory cells are recruited into the joint, where they produce anumber of mediators, including the cytokines interleukin-1 (IL-1), whichcontributes to the chronic sequelae leading to bone and cartilagedestruction, and tumour necrosis factor (TNF-α), which plays a role ininflammation (Dinarello, 1998; Arend and Dayer, 1995; van den Berg,2001). The concentration of IL-1 in plasma is significantly higher inpatients with RA than in healthy individuals and, notably, plasma IL-1levels correlate with RA disease activity (Eastgate et al., 1988).Moreover, synovial fluid levels of IL-1 are correlated with variousradiographic and histologic features of RA (Kahle et al., 1992; Rooneyet al., 1990).

In normal joints, the effects of these and other proinflammatorycytokines are balanced by a variety of anti-inflammatory cytokines andregulatory factors (Burger and Dayer, 1995). The significance of thiscytokine balance is illustrated in juvenile RA patients, who havecyclical increases in fever throughout the day (Prieur et al., 1987).After each peak in fever, a factor that blocks the effects of IL-1 isfound in serum and urine. This factor has been isolated, cloned andidentified as IL-1 receptor antagonist (IL-1ra), a member of the IL-1gene family (Hannum et al., 1990). IL-1ra, as its name indicates, is anatural receptor antagonist that competes with IL-1 for binding to typeI IL-1 receptors and, as a result, blocks the effects of IL-1 (Arend etal., 1998). A 10- to 100-fold excess of IL-1ra may be needed to blockIL-1 effectively; however, synovial cells isolated from patients with RAdo not appear to produce enough IL-1ra to counteract the effects of IL-1(Firestein et al., 1994; Fujikawa et al., 1995).

H. Psoriatic Arthritis

The compounds and methods of this invention may be used for treatingpatients with psoriatic arthritis. Psoriasis is an inflammatory andproliferative skin disorder with a prevalence of 1.5-3%. Approximately20% of patients with psoriasis develop a characteristic form ofarthritis that has several patterns (Gladman, 1992; Jones et al., 1994;Gladman et al., 1995). Some individuals present with joint symptomsfirst but in the majority, skin psoriasis presents first. Aboutone-third of patients have simultaneous exacerbations of their skin andjoint disease (Gladman et al., 1987) and there is a topographicrelationship between nail and distal interphalangeal joint disease(Jones et al., 1994; Wright, 1956). Although the inflammatory processeswhich link skin, nail and joint disease remain elusive, animmune-mediated pathology is implicated.

Psoriatic arthritis (PsA) is a chronic inflammatory arthropathycharacterized by the association of arthritis and psoriasis and wasrecognized as a clinical entity distinct from rheumatoid arthritis (RA)in 1964 (Blumberg et al., 1964). Subsequent studies have revealed thatPsA shares a number of genetic, pathogenic and clinical features withother spondyloarthropathies (SpAs), a group of diseases that compriseankylosing spondylitis, reactive arthritis and enteropathic arthritis(Wright, 1979). The notion that PsA belongs to the SpA group hasrecently gained further support from imaging studies demonstratingwidespread enthesitis in the, including PsA but not RA (McGonagle etal., 1999; McGonagle et al., 1998). More specifically, enthesitis hasbeen postulated to be one of the earliest events occurring in the SpAs,leading to bone remodeling and ankylosis in the spine, as well as toarticular synovitis when the inflamed entheses are close to peripheraljoints. However, the link between enthesitis and the clinicalmanifestations in PsA remains largely unclear, as PsA can present withfairly heterogeneous patterns of joint involvement with variable degreesof severity (Marsal et al., 1999; Salvarani et al., 1998). Thus, otherfactors must be posited to account for the multifarious features of PsA,only a few of which (such as the expression of the HLA-B27 molecule,which is strongly associated with axial disease) have been identified.As a consequence, it remains difficult to map the disease manifestationsto specific pathogenic mechanisms, which means that the treatment ofthis condition remains largely empirical.

Family studies have suggested a genetic contribution to the developmentof PsA (Moll and Wright, 1973). Other chronic inflammatory forms ofarthritis, such as ankylosing spondylitis and rheumatoid arthritis, arethought to have a complex genetic basis. However, the genetic componentof PsA has been difficult to assess for several reasons. There is strongevidence for a genetic predisposition to psoriasis alone that may maskthe genetic factors that are important for the development of PsA.Although most would accept PsA as a distinct disease entity, at timesthere is a phenotypic overlap with rheumatoid arthritis and ankylosingspondylitis. Also, PsA itself is not a homogeneous condition and varioussubgroups have been proposed.

Increased amounts of TNF-α have been reported in both psoriatic skin(Ettehadi et al., 1994) and synovial fluid (Partsch et al., 1997).Recent trials have shown a positive benefit of anti-TNF treatment inboth PsA (Mease et al., 2000) and ankylosing spondylitis (Brandt et al.,2000).

I. Reactive Arthritis

The compounds and methods of this invention may be used for treatingpatients with reactive arthritis. In reactive arthritis (ReA) themechanism of joint damage is unclear, but it is likely that cytokinesplay critical roles. A more prevalent Th1 profile high levels ofinterferon gamma (IFN-γ) and low levels of interleukin 4 (IL-4) has beenreported (Lahesmaa et al., 1992; Schlaak et al., 1992; Simon et al.,1993; Schlaak et al., 1996; Kotake et al., 1999; Ribbens et al., 2000),but several studies have shown relative predominance of IL-4 and IL-10and relative lack of IFN-γ and tumour necrosis factor alpha (TNF-α) inthe synovial membrane (Simon et al., 1994; Yin et al., 1999) and fluid(SF) (Yin et al., 1999; Yin et al., 1997) of reactive arthritis patientscompared with rheumatoid arthritis (RA) patients. A lower level of TNF-αsecretion in reactive arthritis than in RA patients has also beenreported after ex vivo stimulation of peripheral blood mononuclear cells(PBMC) (Braun et al., 1999).

It has been argued that clearance of reactive arthritis-associatedbacteria requires the production of appropriate levels of IFN-γ andTNF-α, while IL-10 acts by suppressing these responses (Autenrieth etal., 1994; Sieper and Braun, 1995). IL-10 is a regulatory cytokine thatinhibits the synthesis of IL-12 and TNF-γ by activated macrophages (deWaal et al., 1991; Hart et al., 1995; Chomarat et al., 1995) and ofIFN-γ by T cells (Macatonia et al., 1993).

J. Enteropathic Arthritis

The compounds and methods of this invention may be used for treatingpatients with enteropathic arthritis. Typically enteropathic arthritis(EA) occurs in combination with inflammatory bowel diseases (IBD) suchas Crohn's disease or ulcerative colitis. It also can affect the spineand sacroiliac joints. Enteropathic arthritis involves the peripheraljoints, usually in the lower extremities such as the knees or ankles. Itcommonly involves only a few or a limited number of joints and mayclosely follow the bowel condition. This occurs in approximately 11% ofpatients with ulcerative colitis and 21% of those with Crohn's disease.The synovitis is generally self-limited and non-deforming.

Enteropathic arthropathies comprise a collection of rheumatologicconditions that share a link to GI pathology. These conditions includereactive (i.e., infection-related) arthritis due to bacteria (e.g.,Shigella, Salmonella, Campylobacter, Yersinia species, Clostridiumdifficile), parasites (e.g., Strongyloides stercoralis, Taenia saginata,Giardia lamblia, Ascaris lumbricoides, Cryptosporidium species), andspondyloarthropathies associated with inflammatory bowel disease (IBD).Other conditions and disorders include intestinal bypass (jejunoileal),arthritis, celiac disease, Whipple disease, and collagenous colitis.

K. Juvenile Rheumatoid Arthritis

The compounds and methods of this invention may be used for treatingpatients with JRA. Juvenile rheumatoid arthritis (JRA), a term for themost prevalent form of arthritis in children, is applied to a family ofillnesses characterized by chronic inflammation and hypertrophy of thesynovial membranes. The term overlaps, but is not completely synonymous,with the family of illnesses referred to as juvenile chronic arthritisand/or juvenile idiopathic arthritis in Europe.

Both innate and adaptive immune systems use multiple cell types, a vastarray of cell surface and secreted proteins, and interconnected networksof positive and negative feedback (Lo et al., 1999). Furthermore, whileseparable in thought, the innate and adaptive wings of the immune systemare functionally intersected (Fearon and Locksley, 1996), and pathologicevents occurring at these intersecting points are likely to be highlyrelevant to our understanding of pathogenesis of adult and childhoodforms of chronic arthritis (Warrington, et al., 2001).

Polyarticular JRA is a distinct clinical subtype characterized byinflammation and synovial proliferation in multiple joints (four ormore), including the small joints of the hands (Jarvis, 2002). Thissubtype of JRA may be severe, because of both its multiple jointinvolvement and its capacity to progress rapidly over time. Althoughclinically distinct, polyarticular JRA is not homogeneous, and patientsvary in disease manifestations, age of onset, prognosis, and therapeuticresponse. These differences very likely reflect a spectrum of variationin the nature of the immune and inflammatory attack that can occur inthis disease (Jarvis, 1998).

L. Early Inflammatory Arthritis

The compounds and methods of this invention may be used for treatingpatients with early inflammatory arthritis. The clinical presentation ofdifferent inflammatory arthropathies is similar early in the course ofdisease. As a result, it is often difficult to distinguish patients whoare at risk of developing the severe and persistent synovitis that leadsto erosive joint damage from those whose arthritis is more self-limited.Such distinction is critical in order to target therapy appropriately,treating aggressively those with erosive disease and avoidingunnecessary toxicity in patients with more self-limited disease. Currentclinical criteria for diagnosing erosive arthropathies such asrheumatoid arthritis (RA) are less effective in early disease andtraditional markers of disease activity such as joint counts and acutephase response do not adequately identify patients likely to have pooroutcomes (Harrison et al., 1998). Parameters reflective of thepathologic events occurring in the synovium are most likely to be ofsignificant prognostic value.

Recent efforts to identify predictors of poor outcome in earlyinflammatory arthritis have identified the presence of RA specificautoantibodies, in particular antibodies towards citrullinated peptides,to be associated with erosive and persistent disease in earlyinflammatory arthritis cohorts. On the basis of this, a cyclicalcitrullinated peptide (CCP) has been developed to assist in theidentification of anti-CCP antibodies in patient sera. Using thisapproach, the presence of anti-CCP antibodies has been shown to bespecific and sensitive for RA, can distinguish RA from otherarthropathies, and can potentially predict persistent, erosive synovitisbefore these outcomes become clinically manifest. Importantly, anti-CCPantibodies are often detectable in sera many years prior to clinicalsymptoms suggesting that they may be reflective of subclinical immuneevents (Nielen et al., 2004; Rantapaa-Dahlqvist et al., 2003).

M. Ankylosing Spondylitis

The compounds and methods of this invention may be used for treatingpatients with ankylosing spondylitis. AS is a disease subset within abroader disease classification of spondyloarthropathy. Patients affectedwith the various subsets of spondyloarthropathy have disease etiologiesthat are often very different, ranging from bacterial infections toinheritance. Yet, in all subgroups, the end result of the diseaseprocess is axial arthritis. Despite the early clinically differencesseen in the various patient populations, many of them end up nearlyidentical after a disease course of ten-to-twenty years. Recent studiessuggest the mean time to clinical diagnosis of ankylosing spondylitisfrom disease onset of disease is 7.5 years (Khan, 1998). These samestudies suggest that the spondyloarthropathies may have prevalence closeto that of rheumatoid arthritis (Feldtkeller et al., 2003; Doran et al.,2003).

AS is a chronic systemic inflammatory rheumatic disorder of the axialskeleton with or without extraskeletal manifestations. Sacroiliac jointsand the spine are primarily affected, but hip and shoulder joints, andless commonly peripheral joints or certain extra-articular structuressuch as the eye, vasculature, nervous system, and gastrointestinalsystem may also be involved. Its etiology is not yet fully understood(Wordsworth, 1995; Calin and Taurog, 1998). It is strongly associatedwith the major histocompatibility class I (MHC I) HLA-B27 allele (Calinand Taurog, 1998). AS affects individuals in the prime of their life andis feared because of its potential to cause chronic pain andirreversible damage of tendons, ligaments, joints, and bones (Brewertonet al., 1973a; Brewerton et al., 1973b; Schlosstein et al., 1973). ASmay occur alone or in association with another form ofspondyloarthropathy such as reactive arthritis, psoriasis, psoriaticarthritis, enthesitis, ulcerative colitis, irritable bowel disease, orCrohn's disease, in which case it is classified as secondary AS.

Typically, the affected sites include the discovertebral, apophyseal,costovertebral, and costotransverse joints of the spine, and theparavertebral ligamentous structures. Inflammation of the entheses,which are sites of musculotendinous and ligamentous attachment to bones,is also prominent in this disease (Calin and Taurog, 1998). The site ofenthesitis is known to be infiltrated by plasma cells, lymphocytes, andpolymorphonuclear cells. The inflammatory process frequently results ingradual fibrous and bony ankylosis, (Ball, 1971; Khan, 1990).

Delayed diagnosis is common because symptoms are often attributed tomore common back problems. A dramatic loss of flexibility in the lumbarspine is an early sign of AS. Other common symptoms include chronic painand stiffness in the lower back which usually starts where the lowerspine is joined to the pelvis, or hip. Although most symptoms begin inthe lumbar and sacroiliac areas, they may involve the neck and upperback as well. Arthritis may also occur in the shoulder, hips and feet.Some patients have eye inflammation, and more severe cases must beobserved for heart valve involvement.

The most frequent presentation is back pain, but disease can beginatypically in peripheral joints, especially in children and women, andrarely with acute iritis (anterior uveitis). Additional early symptomsand signs are diminished chest expansion from diffuse costovertebralinvolvement, low-grade fever, fatigue, anorexia, weight loss, andanemia. Recurrent back pain—often nocturnal and of varying intensity—isan eventual complaint, as is morning stiffness typically relieved byactivity. A flexed or bent-over posture eases back pain and paraspinalmuscle spasm; thus, some degree of kyphosis is common in untreatedpatients.

Systemic manifestations occur in ⅓ of patients. Recurrent, usuallyself-limited, acute iritis (anterior uveitis) rarely is protracted andsevere enough to impair vision. Neurologic signs can occasionally resultfrom compression radiculitis or sciatica, vertebral fracture orsubluxation, and cauda equina syndrome (which consists of impotence,nocturnal urinary incontinence, diminished bladder and rectal sensation,and absence of ankle jerks). Cardiovascular manifestations can includeaortic insufficiency, angina, pericarditis, and ECG conductionabnormalities. A rare pulmonary finding is upper lobe fibrosis,occasionally with cavitation that may be mistaken for TB and can becomplicated by infection with Aspergillus.

AS is characterized by mild or moderate flares of active spondylitisalternating with periods of almost or totally inactive inflammation.Proper treatment in most patients results in minimal or no disabilityand in full, productive lives despite back stiffness. Occasionally, thecourse is severe and progressive, resulting in pronounced incapacitatingdeformities. The prognosis is bleak for patients with refractory iritisand for the rare patient with secondary amyloidosis.

N. Ulcerative Colitis

The compounds and methods of this invention may be used for treatingpatients with ulcerative colitis. Ulcerative colitis is a disease thatcauses inflammation and sores, called ulcers, in the lining of the largeintestine. The inflammation usually occurs in the rectum and lower partof the colon, but it may affect the entire colon. Ulcerative colitisrarely affects the small intestine except for the end section, calledthe terminal ileum. Ulcerative colitis may also be called colitis orproctitis. The inflammation makes the colon empty frequently, causingdiarrhea. Ulcers form in places where the inflammation has killed thecells lining the colon; the ulcers bleed and produce pus.

Ulcerative colitis is an inflammatory bowel disease (IBD), the generalname for diseases that cause inflammation in the small intestine andcolon. Ulcerative colitis can be difficult to diagnose because itssymptoms are similar to other intestinal disorders and to another typeof IBD, Crohn's disease. Crohn's disease differs from ulcerative colitisbecause it causes inflammation deeper within the intestinal wall. Also,Crohn's disease usually occurs in the small intestine, although it canalso occur in the mouth, esophagus, stomach, duodenum, large intestine,appendix, and anus.

Ulcerative colitis may occur in people of any age, but most often itstarts between ages 15 and 30, or less frequently between ages 50 and70. Children and adolescents sometimes develop the disease. Ulcerativecolitis affects men and women equally and appears to run in somefamilies. Theories about what causes ulcerative colitis abound, but nonehave been proven. The most popular theory is that the body's immunesystem reacts to a virus or a bacterium by causing ongoing inflammationin the intestinal wall. People with ulcerative colitis haveabnormalities of the immune system, but doctors do not know whetherthese abnormalities are a cause or a result of the disease. Ulcerativecolitis is not caused by emotional distress or sensitivity to certainfoods or food products, but these factors may trigger symptoms in somepeople.

The most common symptoms of ulcerative colitis are abdominal pain andbloody diarrhea. Patients also may experience fatigue, weight loss, lossof appetite, rectal bleeding, and loss of body fluids and nutrients.About half of patients have mild symptoms. Others suffer frequent fever,bloody diarrhea, nausea, and severe abdominal cramps. Ulcerative colitismay also cause problems such as arthritis, inflammation of the eye,liver disease (hepatitis, cirrhosis, and primary sclerosingcholangitis), osteoporosis, skin rashes, and anemia. No one knows forsure why problems occur outside the colon. Scientists think thesecomplications may occur when the immune system triggers inflammation inother parts of the body. Some of these problems go away when the colitisis treated.

A thorough physical exam and a series of tests may be required todiagnose ulcerative colitis. Blood tests may be done to check foranemia, which could indicate bleeding in the colon or rectum. Bloodtests may also uncover a high white blood cell count, which is a sign ofinflammation somewhere in the body. By testing a stool sample, thedoctor can detect bleeding or infection in the colon or rectum. Thedoctor may do a colonoscopy or sigmoidoscopy. For either test, thedoctor inserts an endoscope—a long, flexible, lighted tube connected toa computer and TV monitor—into the anus to see the inside of the colonand rectum. The doctor will be able to see any inflammation, bleeding,or ulcers on the colon wall. During the exam, the doctor may do abiopsy, which involves taking a sample of tissue from the lining of thecolon to view with a microscope. A barium enema x ray of the colon mayalso be required. This procedure involves filling the colon with barium,a chalky white solution. The barium shows up white on x-ray film,allowing the doctor a clear view of the colon, including any ulcers orother abnormalities that might be there.

Treatment for ulcerative colitis depends on the seriousness of thedisease. Most people are treated with medication. In severe cases, apatient may need surgery to remove the diseased colon. Surgery is theonly cure for ulcerative colitis. Some people whose symptoms aretriggered by certain foods are able to control the symptoms by avoidingfoods that upset their intestines, like highly seasoned foods, rawfruits and vegetables, or milk sugar (lactose). Each person mayexperience ulcerative colitis differently, so treatment is adjusted foreach individual. Emotional and psychological support is important. Somepeople have remissions—periods when the symptoms go away—that last formonths or even years. However, most patients' symptoms eventuallyreturn. This changing pattern of the disease means one cannot alwaystell when a treatment has helped. Some people with ulcerative colitismay need medical care for some time, with regular doctor visits tomonitor the condition.

O. Crohn's Disease

The compounds and methods of this invention may be used for treatingpatients with Crohn's disease. Another disorder for whichimmunosuppression has been tried is Crohn's disease. Crohn's diseasesymptoms include intestinal inflammation and the development ofintestinal stenosis and fistulas; neuropathy often accompanies thesesymptoms. Anti-inflammatory drugs, such as 5-aminosalicylates (e.g.,mesalamine) or corticosteroids, are typically prescribed, but are notalways effective (reviewed in Botoman et al., 1998). Immunosuppressionwith cyclosporine is sometimes beneficial for patients resistant to orintolerant of corticosteroids (Brynskov et al., 1989).

Efforts to develop diagnostic and treatment tools against Crohn'sdisease have focused on the central role of cytokines (Schreiber, 1998;van Hogezand and Verspaget, 1998). Cytokines are small secreted proteinsor factors (5 to 20 kD) that have specific effects on cell-to-cellinteractions, intercellular communication, or the behavior of othercells. Cytokines are produced by lymphocytes, especially T_(H)1 andT_(H)2 lymphocytes, monocytes, intestinal macrophages, granulocytes,epithelial cells, and fibroblasts (reviewed in Rogler and. Andus, 1998;Galley and Webster, 1996). Some cytokines are pro-inflammatory (e.g.,TNF-α, IL-1(α and β), IL-6, IL-8, IL-12, or leukemia inhibitory factor[LIF]); others are anti-inflammatory (e.g., IL-1 receptor antagonist,IL-4, IL-10, IL-11, and TGF-β). However, there may be overlap andfunctional redundancy in their effects under certain inflammatoryconditions.

In active cases of Crohn's disease, elevated concentrations of TNF-α andIL-6 are secreted into the blood circulation, and TNF-α, IL-1, IL-6, andIL-8 are produced in excess locally by mucosal cells (id.; Funakoshi etal., 1998). These cytokines can have far-ranging effects onphysiological systems including bone development, hematopoiesis, andliver, thyroid, and neuropsychiatric function. Also, an imbalance of theIL-1β/IL-1ra ratio, in favor of pro-inflammatory IL-1β, has beenobserved in patients with Crohn's disease (Rogler and Andus, 1998; Saikiet al., 1998; Dionne et al., 1998; but see Kuboyama, 1998). One studysuggested that cytokine profiles in stool samples could be a usefuldiagnostic tool for Crohn's disease (Saiki et al., 1998).

Treatments that have been proposed for Crohn's disease include the useof various cytokine antagonists (e.g., IL-1ra), inhibitors (e.g., ofIL-1β converting enzyme and antioxidants) and anti-cytokine antibodies(Rogler and Andus, 1998; van Hogezand and Verspaget, 1998; Reimund etal., 1998; Lugering et al., 1998; McAlindon et al., 1998). Inparticular, monoclonal antibodies against TNF-α have been tried withsome success in the treatment of Crohn's disease (Targan et al., 1997;Stack et al., 1997; van Dullemen et al., 1995). These compounds may beused in combination therapy with compounds of the present disclosure.

Another approach to the treatment of Crohn's disease has focused on atleast partially eradicating the bacterial community that may betriggering the inflammatory response and replacing it with anon-pathogenic community. For example, U.S. Pat. No. 5,599,795 disclosesa method for the prevention and treatment of Crohn's disease in humanpatients. Their method was directed to sterilizing the intestinal tractwith at least one antibiotic and at least one anti-fungal agent to killoff the existing flora and replacing them with different, select,well-characterized bacteria taken from normal humans. Borody taught amethod of treating Crohn's disease by at least partial removal of theexisting intestinal microflora by lavage and replacement with a newbacterial community introduced by fecal inoculum from a disease-screenedhuman donor or by a composition comprising Bacteroides and Escherichiacoli species. (U.S. Pat. No. 5,443,826).

P. Systemic Lupus Erythematosus

The compounds and methods of this invention may be used for treatingpatients with SLE. There has also been no known cause for autoimmunediseases such as systemic lupus erythematosus. Systemic lupuserythematosus (SLE) is an autoimmune rheumatic disease characterized bydeposition in tissues of autoantibodies and immune complexes leading totissue injury (Kotzin, 1996). In contrast to autoimmune diseases such asMS and type 1 diabetes mellitus, SLE potentially involves multiple organsystems directly, and its clinical manifestations are diverse andvariable (reviewed by Kotzin and O'Dell, 1995). For example, somepatients may demonstrate primarily skin rash and joint pain, showspontaneous remissions, and require little medication. At the other endof the spectrum are patients who demonstrate severe and progressivekidney involvement that requires therapy with high doses of steroids andcytotoxic drugs such as cyclophosphamide (Kotzin, 1996).

The serological hallmark of SLE, and the primary diagnostic testavailable, is elevated serum levels of IgG antibodies to constituents ofthe cell nucleus, such as double-stranded DNA (dsDNA), single-strandedDNA (ss-DNA), and chromatin. Among these autoantibodies, IgG anti-dsDNAantibodies play a major role in the development of lupusglomerulonephritis (G N) (Hahn and Tsao, 1993; Ohnishi et al., 1994).Glomerulonephritis is a serious condition in which the capillary wallsof the kidney's blood purifying glomeruli become thickened by accretionson the epithelial side of glomerular basement membranes. The disease isoften chronic and progressive and may lead to eventual renal failure.

Q. Irritable Bowel Syndrome

The compounds and methods of this invention may be used for treatingpatients with Irritable bowel syndrome (IBS). IBS is a functionaldisorder characterized by abdominal pain and altered bowel habits. Thissyndrome may begin in young adulthood and can be associated withsignificant disability. This syndrome is not a homogeneous disorder.Rather, subtypes of IBS have been described on the basis of thepredominant symptom—diarrhea, constipation, or pain. In the absence of“alarm” symptoms, such as fever, weight loss, and gastrointestinalbleeding, a limited workup is needed. Once a diagnosis of IBS is made,an integrated treatment approach can effectively reduce the severity ofsymptoms. IBS is a common disorder, although its prevalence rates havevaried. In general, IBS affects about 15% of US adults and occurs aboutthree times more often in women than in men (Jailwala et al., 2000).

IBS accounts for between 2.4 million and 3.5 million visits tophysicians each year. It not only is the most common condition seen bygastroenterologists but also is one of the most common gastrointestinalconditions seen by primary care physicians (Everhart et al., 1991;Sandler, 1990).

IBS is also a costly disorder. Compared with persons who do not havebowel symptoms, persons with IBS miss three times as many workdays andare more likely to report being too sick to work (Drossman et al., 1993;Drossman et al., 1997). Moreover, those with IBS incur hundreds ofdollars more in medical charges than persons without bowel disorders(Talley et al., 1995).

No specific abnormality accounts for the exacerbations and remissions ofabdominal pain and altered bowel habits experienced by patients withIBS. The evolving theory of IBS suggests dysregulation at multiplelevels of the brain-gut axis. Dysmotility, visceral hypersensitivity,abnormal modulation of the central nervous system (CNS), and infectionhave all been implicated. In addition, psychosocial factors play animportant modifying role. Abnormal intestinal motility has long beenconsidered a factor in the pathogenesis of IBS. Transit time through thesmall intestine after a meal has been shown to be shorter in patientswith diarrhea-predominant IBS than in patients who have theconstipation-predominant or pain-predominant subtype (Cann et al.,1983).

In studies of the small intestine during fasting, the presence of bothdiscrete, clustered contractions and prolonged, propagated contractionshas been reported in patients with IBS (Kellow and Phillips, 1987). Theyalso experience pain with irregular contractions more often than healthypersons (Kellow and Phillips, 1987; Horwitz and Fisher, 2001)

These motility findings do not account for the entire symptom complex inpatients with IBS; in fact, most of these patients do not havedemonstrable abnormalities (Rothstein, 2000). Patients with IBS haveincreased sensitivity to visceral pain. Studies involving balloondistention of the rectosigmoid colon have shown that patients with IBSexperience pain and bloating at pressures and volumes much lower thancontrol subjects (Whitehead et al., 1990). These patients maintainnormal perception of somatic stimuli.

Multiple theories have been proposed to explain this phenomenon. Forexample, receptors in the viscera may have increased sensitivity inresponse to distention or intraluminal contents. Neurons in the dorsalhorn of the spinal cord may have increased excitability. In addition,alteration in CNS processing of sensations may be involved (Drossman etal., 1997). Functional magnetic resonance imaging studies have recentlyshown that compared with control subjects, patients with IBS haveincreased activation of the anterior cingulate cortex, an important paincenter, in response to a painful rectal stimulus (Mertz et al., 2000).

Increasingly, evidence suggests a relationship between infectiousenteritis and subsequent development of IBS. Inflammatory cytokines mayplay a role. In a survey of patients with a history of confirmedbacterial gastroenteritis (Neal et al., 1997), 25% reported persistentalteration of bowel habits. Persistence of symptoms may be due topsychological stress at the time of acute infection (Gwee et al., 1999).

Recent data suggest that bacterial overgrowth in the small intestine mayhave a role in IBS symptoms. In one study (Pimentel et al., 2000), 157(78%) of 202 IBS patients referred for hydrogen breath testing had testfindings that were positive for bacterial overgrowth. Of the 47 subjectswho had follow-up testing, 25 (53%) reported improvement in symptoms(i.e., abdominal pain and diarrhea) with antibiotic treatment.

IBS may present with a range of symptoms. However, abdominal pain andaltered bowel habits remain the primary features. Abdominal discomfortis often described as crampy in nature and located in the left lowerquadrant, although the severity and location can differ greatly.Patients may report diarrhea, constipation, or alternating episodes ofdiarrhea and constipation. Diarrheal symptoms are typically described assmall-volume, loose stools, and stool is sometimes accompanied by mucusdischarge. Patients also may report bloating, fecal urgency, incompleteevacuation, and abdominal distention. Upper gastrointestinal symptoms,such as gastroesophageal reflux, dyspepsia, or nausea, may also bepresent (Lynn and Friedman, 1993).

Persistence of symptoms is not an indication for further testing; it isa characteristic of IBS and is itself an expected symptom of thesyndrome. More extensive diagnostic evaluation is indicated in patientswhose symptoms are worsening or changing. Indications for furthertesting also include presence of alarm symptoms, onset of symptoms afterage 50, and a family history of colon cancer. Tests may includecolonoscopy, computed tomography of the abdomen and pelvis, and bariumstudies of the small or large intestine.

R. Sjögren's Syndrome

The compounds and methods of this invention may be used for treatingpatients with SS. Primary Sjögren's syndrome (SS) is a chronic, slowlyprogressive, systemic autoimmune disease, which affects predominantlymiddle-aged women (female-to-male ratio 9:1), although it can be seen inall ages including childhood (Jonsson et al., 2002). It is characterizedby lymphocytic infiltration and destruction of the exocrine glands,which are infiltrated by mononuclear cells including CD4+, CD8+lymphocytes and B-cells (Jonsson et al., 2002). In addition,extraglandular (systemic) manifestations are seen in one-third ofpatients (Jonsson et al., 2001).

The glandular lymphocytic infiltration is a progressive feature (Jonssonet al., 1993), which, when extensive, may replace large portions of theorgans. Interestingly, the glandular infiltrates in some patientsclosely resemble ectopic lymphoid microstructures in the salivary glands(denoted as ectopic germinal centers) (Salomonsson et al., 2002; Xanthouet al., 2001). In SS, ectopic GCs are defined as T and B cell aggregatesof proliferating cells with a network of follicular dendritic cells andactivated endothelial cells. These GC-like structures formed within thetarget tissue also portray functional properties with production ofautoantibodies (anti-Ro/SSA and anti-La/SSB) (Salomonsson and Jonsson,2003).

In other systemic autoimmune diseases, such as RA, factors critical forectopic GCs have been identified. Rheumatoid synovial tissues with GCswere shown to produce chemokines CXCL13, CCL21 and lymphotoxin (LT)-β(detected on follicular center and mantle zone B cells). Multivariateregression analysis of these analytes identified CXCL13 and LT-β as thesolitary cytokines predicting GCs in rheumatoid synovitis (Weyand andGoronzy, 2003). Recently CXCL13 and CXCR5 in salivary glands has beenshown to play an essential role in the inflammatory process byrecruiting B and T cells, therefore contributing to lymphoid neogenesisand ectopic GC formation in SS (Salomonsson et al., 2002).

S. Psoriasis

The compounds and methods of this invention may be used for treatingpatients with psoriasis. Psoriasis is a chronic skin disease of scalingand inflammation that affects 2 to 2.6 percent of the United Statespopulation, or between 5.8 and 7.5 million people. Although the diseaseoccurs in all age groups, it primarily affects adults. It appears aboutequally in males and females. Psoriasis occurs when skin cells quicklyrise from their origin below the surface of the skin and pile up on thesurface before they have a chance to mature. Usually this movement (alsocalled turnover) takes about a month, but in psoriasis it may occur inonly a few days. In its typical form, psoriasis results in patches ofthick, red (inflamed) skin covered with silvery scales. These patches,which are sometimes referred to as plaques, usually itch or feel sore.They most often occur on the elbows, knees, other parts of the legs,scalp, lower back, face, palms, and soles of the feet, but they canoccur on skin anywhere on the body. The disease may also affect thefingernails, the toenails, and the soft tissues of the genitals andinside the mouth. While it is not unusual for the skin around affectedjoints to crack, approximately 1 million people with psoriasisexperience joint inflammation that produces symptoms of arthritis. Thiscondition is called psoriatic arthritis.

Psoriasis is a skin disorder driven by the immune system, especiallyinvolving a type of white blood cell called a T cell. Normally, T cellshelp protect the body against infection and disease. In the case ofpsoriasis, T cells are put into action by mistake and become so activethat they trigger other immune responses, which lead to inflammation andto rapid turnover of skin cells. In about one-third of the cases, thereis a family history of psoriasis. Researchers have studied a largenumber of families affected by psoriasis and identified genes linked tothe disease. People with psoriasis may notice that there are times whentheir skin worsens, then improves. Conditions that may cause flareupsinclude infections, stress, and changes in climate that dry the skin.Also, certain medicines, including lithium and beta blockers, which areprescribed for high blood pressure, may trigger an outbreak or worsenthe disease.

T. Infectious Diseases

Compounds of the present disclosure may be useful in the treatment ofinfectious diseases, including viral and bacterial infections. As notedabove, such infections may be associated with severe localized orsystemic inflammatory responses. For example, influenza may cause severeinflammation of the lung and bacterial infection can cause the systemichyperinflammatory response, including the excessive production ofmultiple inflammatory cytokines, that is the hallmark of sepsis. Inaddition, compounds of the invention may be useful in directlyinhibiting the replication of viral pathogens. Previous studies havedemonstrated that related compounds such as CDDO can inhibit thereplication of HIV in macrophages (Vazquez et al., 2005). Other studieshave indicated that inhibition of NF-kappa B signaling may inhibitinfluenza virus replication, and that cyclopentenone prostaglandins mayinhibit viral replication (e.g., Mazur et al., 2007; Pica et al., 2000).

VI. PHARMACEUTICAL FORMULATIONS AND ROUTES OF ADMINISTRATION

The compounds of the present disclosure may be administered by a varietyof methods, e.g., orally or by injection (e.g. subcutaneous,intravenous, intraperitoneal, etc.). Depending on the route ofadministration, the active compounds may be coated in a material toprotect the compound from the action of acids and other naturalconditions which may inactivate the compound. They may also beadministered by continuous perfusion/infusion of a disease or woundsite.

To administer the therapeutic compound by other than parenteraladministration, it may be necessary to coat the compound with, orco-administer the compound with, a material to prevent its inactivation.For example, the therapeutic compound may be administered to a patientin an appropriate carrier, for example, liposomes, or a diluent.Pharmaceutically acceptable diluents include saline and aqueous buffersolutions. Liposomes include water-in-oil-in-water CGF emulsions as wellas conventional liposomes (Strejan et al., 1984).

The therapeutic compound may also be administered parenterally,intraperitoneally, intraspinally, or intracerebrally. Dispersions can beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations may contain a preservative to prevent the growth ofmicroorganisms.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. In all cases, the composition must be sterileand must be fluid to the extent that easy syringability exists. It mustbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (such as, glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and vegetable oils. The proper fluidity canbe maintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, sodium chloride, orpolyalcohols such as mannitol and sorbitol, in the composition.Prolonged absorption of the injectable compositions can be brought aboutby including in the composition an agent which delays absorption, forexample, aluminum monostearate or gelatin.

Sterile injectable solutions can be prepared by incorporating thetherapeutic compound in the required amount in an appropriate solventwith one or a combination of ingredients enumerated above, as required,followed by filtered sterilization. Generally, dispersions are preparedby incorporating the therapeutic compound into a sterile carrier whichcontains a basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and freeze-drying which yields a powder ofthe active ingredient (i.e., the therapeutic compound) plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The therapeutic compound can be orally administered, for example, withan inert diluent or an assimilable edible carrier. The therapeuticcompound and other ingredients may also be enclosed in a hard or softshell gelatin capsule, compressed into tablets, or incorporated directlyinto the subject's diet. For oral therapeutic administration, thetherapeutic compound may be incorporated with excipients and used in theform of ingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. The percentage of thetherapeutic compound in the compositions and preparations may, ofcourse, be varied. The amount of the therapeutic compound in suchtherapeutically useful compositions is such that a suitable dosage willbe obtained.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subjects to be treated; each unitcontaining a predetermined quantity of therapeutic compound calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical carrier. The specification for the dosage unitforms of the invention are dictated by and directly dependent on (a) theunique characteristics of the therapeutic compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such a therapeutic compound for the treatment ofa selected condition in a patient.

The therapeutic compound may also be administered topically to the skin,eye, or mucosa. Alternatively, if local delivery to the lungs is desiredthe therapeutic compound may be administered by inhalation in adry-powder or aerosol formulation.

Active compounds are administered at a therapeutically effective dosagesufficient to treat a condition associated with a condition in apatient. A “therapeutically effective amount” preferably reduces theamount of symptoms of the condition in the infected patient by at leastabout 20%, more preferably by at least about 40%, even more preferablyby at least about 60%, and still more preferably by at least about 80%relative to untreated subjects. For example, the efficacy of a compoundcan be evaluated in an animal model system that may be predictive ofefficacy in treating the disease in humans, such as the model systemsshown in the examples and drawings.

The actual dosage amount of a compound of the present disclosure orcomposition comprising a compound of the present disclosure administeredto a subject may be determined by physical and physiological factorssuch as age, sex, body weight, severity of condition, the type ofdisease being treated, previous or concurrent therapeutic interventions,idiopathy of the subject and on the route of administration. Thesefactors may be determined by a skilled artisan. The practitionerresponsible for administration will typically determine theconcentration of active ingredient(s) in a composition and appropriatedose(s) for the individual subject. The dosage may be adjusted by theindividual physician in the event of any complication.

An effective amount typically will vary from about 0.001 mg/kg to about1,000 mg/kg, from about 0.01 mg/kg to about 750 mg/kg, from about 100mg/kg to about 500 mg/kg, from about 1.0 mg/kg to about 250 mg/kg, fromabout 10.0 mg/kg to about 150 mg/kg in one or more dose administrationsdaily, for one or several days (depending, of course, of the mode ofadministration and the factors discussed above). Other suitable doseranges include 1 mg to 10,000 mg per day, 100 mg to 10,000 mg per day,500 mg to 10,000 mg per day, and 500 mg to 1,000 mg per day. In someparticular embodiments, the amount is less than 10,000 mg per day with arange, for example, of 750 mg to 9,000 mg per day.

The effective amount may be less than 1 mg/kg/day, less than 500mg/kg/day, less than 250 mg/kg/day, less than 100 mg/kg/day, less than50 mg/kg/day, less than 25 mg/kg/day or less than 10 mg/kg/day. It mayalternatively be in the range of 1 mg/kg/day to 200 mg/kg/day. Forexample, regarding treatment of diabetic patients, the unit dosage maybe an amount that reduces blood glucose by at least 40% as compared toan untreated subject. In another embodiment, the unit dosage is anamount that reduces blood glucose to a level that is ±10% of the bloodglucose level of a non-diabetic subject.

In other non-limiting examples, a dose may also comprise from about 1microgram/kg/body weight, about 5 microgram/kg/body weight, about 10micro-gram/kg/body weight, about 50 microgram/kg/body weight, about 100micro-gram/kg/body weight, about 200 microgram/kg/body weight, about 350micro-gram/kg/body weight, about 500 microgram/kg/body weight, about 1milligram/kg/body weight, about 5 milligram/kg/body weight, about 10milligram/kg/body weight, about 50 milligram/kg/body weight, about 100milligram/kg/body weight, about 200 milli-gram/kg/body weight, about 350milligram/kg/body weight, about 500 milligram/kg/body weight, to about1,000 mg/kg/body weight or more per administration, and any rangederivable therein. In non-limiting examples of a derivable range fromthe numbers listed herein, a range of about 5 mg/kg/body weight to about100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500milligram/kg/body weight, etc., can be administered, based on thenumbers described above.

In certain embodiments, a pharmaceutical composition of the presentdisclosure may comprise, for example, at least about 0.1% of a compoundof the present disclosure. In other embodiments, the compound of thepresent disclosure may comprise between about 2% to about 75% of theweight of the unit, or between about 25% to about 60%, for example, andany range derivable therein.

Single or multiple doses of the agents are contemplated. Desired timeintervals for delivery of multiple doses can be determined by one ofordinary skill in the art employing no more than routineexperimentation. As an example, subjects may be administered two dosesdaily at approximately 12 hour intervals. In some embodiments, the agentis administered once a day.

The agent(s) may be administered on a routine schedule. As used herein aroutine schedule refers to a predetermined designated period of time.The routine schedule may encompass periods of time which are identicalor which differ in length, as long as the schedule is predetermined. Forinstance, the routine schedule may involve administration twice a day,every day, every two days, every three days, every four days, every fivedays, every six days, a weekly basis, a monthly basis or any set numberof days or weeks there-between. Alternatively, the predetermined routineschedule may involve administration on a twice daily basis for the firstweek, followed by a daily basis for several months, etc. In otherembodiments, the invention provides that the agent(s) may taken orallyand that the timing of which is or is not dependent upon food intake.Thus, for example, the agent can be taken every morning and/or everyevening, regardless of when the subject has eaten or will eat.

VII. COMBINATION THERAPY

In addition to being used as a monotherapy, the compounds of the presentdisclosure may also find use in combination therapies. Effectivecombination therapy may be achieved with a single composition orpharmacological formulation that includes both agents, or with twodistinct compositions or formulations, at the same time, wherein onecomposition includes a compound of this invention, and the otherincludes the second agent(s). Alternatively, the therapy may precede orfollow the other agent treatment by intervals ranging from minutes tomonths.

Various combinations may be employed, such as when a compound of thepresent disclosure is “A” and “B” represents a secondary agent,non-limiting examples of which are described below:

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B B/B/B/A B/B/A/BA/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/AA/A/B/A

Administration of the compounds of the present disclosure to a patientwill follow general protocols for the administration of pharmaceuticals,taking into account the toxicity, if any, of the drug. It is expectedthat the treatment cycles would be repeated as necessary.

Beta interferons may be suitable secondary agents. These are medicationsderived from human cytokines which help regulate the immune system. Theyinclude interferon β-1b and interferon β-1a. Betaseron has been approvedby the FDA for relapsing forms of secondary progressive MS. Furthermore,the FDA has approved the use of several β-interferons as treatments forpeople who have experienced a single attack that suggests multiplesclerosis, and who may be at risk of future attacks and developingdefinite MS. For example, risk of MS may be suggested when an MRI scanof the brain shows lesions that predict a high risk of conversion todefinite MS.

Glatiramer acetate is a further example of a secondary agent that may beused in a combination treatment. Glatiramer is presently used to treatrelapsing remitting MS. It is made of four amino acids that are found inmyelin. This drug is reported to stimulate T cells in the body's immunesystem to change from harmful, pro-inflammatory agents to beneficial,anti-inflammatory agents that work to reduce inflammation at lesionsites.

Another potential secondary agent is mitoxantrone, a chemotherapy drugused for many cancers. This drug is also FDA-approved for treatment ofaggressive forms of relapsing remitting MS, as well as certain forms ofprogressive MS. It is given intravenously, typically every three months.This medication is effective, but is limited by cardiac toxicity.Novantrone has been approved by the FDA for secondary progressive,progressive-relapsing, and worsening relapsing-remitting MS.

Another potential secondary agent is natalizumab. In general,natalizumab works by blocking the attachment of immune cells to brainblood vessels, which is a necessary step for immune cells to cross intothe brain, thus reducing the immune cells' inflammatory action on brainneurons. Natalizumab has been shown to significantly reduce thefrequency of attacks in people with relapsing MS.

In the case of relapsing remitting MS, patients may be given intravenouscorticosteroids, such as methylprednisolone, as a secondary agent, toend the attack sooner and leave fewer lasting deficits.

Other common drugs for MS that may be used in combination with thepresent oleanolic acid derivatives include immunosuppressive drugs suchas azathioprine, cladribine and cyclophosphamide.

It is contemplated that other anti-inflammatory agents may be used inconjunction with the treatments of the current invention. Other COXinhibitors may be used, including arylcarboxylic acids (salicylic acid,acetylsalicylic acid, diflunisal, choline magnesium trisalicylate,salicylate, benorylate, flufenamic acid, mefenamic acid, meclofenamicacid and triflumic acid), arylalkanoic acids (diclofenac, fenclofenac,alclofenac, fentiazac, ibuprofen, flurbiprofen, ketoprofen, naproxen,fenoprofen, fenbufen, suprofen, indoprofen, tiaprofenic acid,benoxaprofen, pirprofen, tolmetin, zomepirac, clopinac, indomethacin andsulindac) and enolic acids (phenylbutazone, oxyphenbutazone,azapropazone, feprazone, piroxicam, and isoxicam. See also U.S. Pat. No.6,025,395, which is incorporated herein by reference.

Histamine H2 receptor blocking agents may also be used in conjunctionwith the compounds of the current invention, including cimetidine,ranitidine, famotidine and nizatidine.

Treatment with acetylcholinesterase inhibitors such as tacrine,donepizil, metrifonate and rivastigmine for the treatment of Alzheimer'sand other disease in conjunction with the compounds of the presentdisclosure is contemplated. Other acetylcholinesterase inhibitors may bedeveloped which may be used once approved include rivastigmine andmetrifonate. Acetylcholinesterase inhibitors increase the amount ofneurotransmitter acetylcholine at the nerve terminal by decreasing itsbreakdown by the enzyme cholinesterase.

MAO-B inhibitors such as selegilene may be used in conjunction with thecompounds of the current invention. Selegilene is used for Parkinson'sdisease and irreversibly inhibits monoamine oxidase type B (MAO-B).Monoamine oxidase is an enzyme that inactivates the monoamineneurotransmitters norepinephrine, serotonin and dopamine.

Dietary and nutritional supplements with reported benefits for treatmentor prevention of Parkinson's, Alzheimer's, multiple sclerosis,amyotrophic lateral sclerosis, rheumatoid arthritis, inflammatory boweldisease, and all other diseases whose pathogenesis is believed toinvolve excessive production of either nitric oxide (NO) orprostaglandins, such as acetyl-L-carnitine, octacosanol, eveningprimrose oil, vitamin B6, tyrosine, phenylalanine, vitamin C, L-dopa, ora combination of several antioxidants may be used in conjunction withthe compounds of the current invention.

For the treatment or prevention of cancer, compounds of the inventionmay be combined with one or more of the following: radiation,chemotherapy agents (e.g., cytotoxic agents such as anthracyclines,vincristine, vinblastin, microtubule-targeting agents such as paclitaxeland docetaxel, 5-FU and related agents, cisplatin and otherplatinum-containing compounds, irinotecan and topotecan, gemcitabine,temozolomide, etc.), targeted therapies (e.g., imatinib, bortezomib,bevacizumab, rituximab), or vaccine therapies designed to promote anenhanced immune response targeting cancer cells.

For the treatment or prevention of autoimmune disease, compounds of theinvention may be combined with one or more of the following:corticosteroids, methotrexate, anti-TNF antibodies, other TNF-targetingprotein therapies, and NSAIDs. For the treatment of prevention ofcardiovascular diseases, compounds of the invention may be combined withantithrombotic therapies, anticholesterol therapies such as statins(e.g., atorvastatin), and surgical interventions such as stenting orcoronary artery bypass grafting. For the treatment of osteoporosis,compounds of the invention may be combined with antiresorptive agentssuch as bisphosphonates or anabolic therapies such as teriparatide orparathyroid hormone. For the treatment of neuropsychiatric conditions,compounds of the invention may be combined with antidepressants (e.g.,imipramine or SSRIs such as fluoxetine), antipsychotic agents (e.g.,olanzapine, sertindole, risperidone), mood stabilizers (e.g., lithium,valproate semisodium), or other standard agents such as anxiolyticagents. For the treatment of neurological disorders, compounds of theinvention may be combined with anticonvulsant agents (e.g., valproatesemisodium, gabapentin, phenyloin, carbamazepine, and topiramate),antithrombotic agents (e.g., tissue plasminogen activator), oranalgesics (e.g., opioids, sodium channel blockers, and otherantinociceptive agents).

For the treatment of disorders involving oxidative stress, compounds ofthe present disclosure may be combined with tetrahydrobiopterin (BH4) orrelated compounds. BH4 is a cofactor for constitutive forms of nitricoxide synthase, and may be depleted by reactions with peroxynitrite.Peroxynitrite is formed by the reaction of nitric oxide and superoxide.Thus, under conditions of oxidative stress excessive levels ofsuperoxide can deplete normal, beneficial levels of nitric oxide byconverting NO to peroxynitrite. The resulting depletion of BH4 byreaction with peroxynitrite results in the “uncoupling” of nitric oxidesynthases so that they form superoxide rather than NO. This adds to theoversupply of superoxide and prolongs the depletion of NO. Addition ofexogenous BH4 can reverse this uncoupling phenomenon, restoring theproduction of NO and reducing the level of oxidative stress in tissues.This mechanism is expected to complement the actions of compounds of theinvention, which reduce oxidative stress by other means, as discussedabove and throughout this invention.

VIII. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Methods and Materials

Nitric Oxide production and cell viability. RAW264.7 macrophages werepre-treated with DMSO or drugs for 2 hours, then treated withrecombinant mouse IFNγ (Sigma) for 24 hours. NO concentration in mediawas determined using the Griess reagent system (Promega). Cell viabilitywas determined using WST-1 reagent (Roche).

STAT3 phosphorylation. HeLa cells were treated with the indicatedcompounds and concentrations for 6 hours and subsequently stimulatedwith 20 ng/ml recombinant human IL-6 (R&D Systems) for 15 minutes.Lysates were immunoblotted with antibodies against phosphorylated ortotal STAT3 (Cell Signaling).

NF-κB activation. HeLa cells were transfected with pNF-κB-Luc(inducible, Stratagene) and pRL-TK (constitutive, Promega) reporterplasmids. Twenty-four hours later cells were pre-treated with theindicated compounds for 2 hours. DMSO served as a vehicle control.Following pre-treatment, cells were stimulated with 20 ng/ml recombinanthuman TNFα (BD Biosciences) for 3 hours. Reporter activity was measuredusing DualGlo luciferase reporter system (Promega) and pNF-κB luciferaseactivity was normalized against pRL-TK luciferase activity.Fold-induction of mean luciferase activity relative to unstimulated(-TNFα) samples is shown. Error bars represent the SD of the mean of 6samples.

IκBα degradation. HeLa cells were treated with indicated compounds andconcentrations for 6 hours and subsequently stimulated with 20 ng/mlTNFα for 15 minutes. Lysates were blotted with antibodies against IκBα(Santa Cruz) and actin (Chemicon).

COX-2 induction Western blot. RAW264.7 cells were pre-treated for 2hours with indicated compounds and subsequently stimulated with 10 ng/mlIFNγ for an additional 24 hours. COX-2 protein levels were assayed byimmunoblotting using an antibody from Santa Cruz. Actin was used as aloading control.

Nrf2 target gene induction. MDA-MB-435 human melanoma cells were treatedwith vehicle (DMSO) or the indicated compounds and concentrations for 16hours. HO-1, thioredoxin reductase-1 (TrxR1), γ-glutamylcysteinesynthetase (γ-GCS), and ferritin heavy chain mRNA levels were quantifiedusing qPCR and were normalized relative to a DMSO-treated sample run inparallel. Values are averages of duplicate wells. Primer sequences areas follows.

HO-1 FW: TCCGATGGGTCCTTACACTC, (SEQ ID NO: 1) HO-1 REV:TAGGCTCCTTCCTCCTTTCC, (SEQ ID NO: 2) TrxR1 FW: GCAGCACTGAGTGGTCAAAA,(SEQ ID NO: 3) TrxR1 REV: GGTCAACTGCCTCAATTGCT, (SEQ ID NO: 4) γ-GCS FW:GCTGTGGCTACTGCGGTATT, (SEQ ID NO: 5) γ-GCS REV ATCTGCCTCAATGACACCAT,(SEQ ID NO: 6) Ferritin HC FW: ATGAGCAGGTGAAAGCCATC, (SEQ ID NO: 7)Ferritin HC REV: TAAAGGAAACCCCAACATGC, (SEQ ID NO: 8) S9 FW:GATTACATCCTGGGCCTGAA, (SEQ ID NO: 9) S9 REV: GAGCGCAGAGAGAAGTCGAT.(SEQ ID NO: 10)

Comparison Compounds. Some of the experimental results presented belowand throughout this application present data for not only the compoundsdiscussed above, but also for one or more of the triterpenoidderivatives shown in the table below.

Several of the above compounds, including 401, 402, 402-56 and 402 canbe prepared according to the methods taught by Honda et al. (1998),Honda et al. (2000b), Honda et al. (2002), Yates et al. (2007), U.S.Pat. No. 6,974,801, and U.S. Provisional Applications 61/046,342,61/046,352, 61/046,366, 61/111,269, and 61/111,294, which are allincorporated herein by reference. The synthesis of the other compoundsmay be prepared according to the methods disclosed in one or more of thefollowing applications filed concurrently herewith, each of which isincorporated herein by reference in their entireties: U.S. patentapplication by Eric Anderson, Xin Jiang and Melean Visnick, entitled“Antioxidant Inflammation Modulators: Oleanolic Acid Derivatives withAmino and Other Modifications At C-17,” filed Apr. 20, 2009; U.S. patentapplication by Xin Jiang, Jack Greiner, Lester Maravetz, Stephen S.Szucs, Melean Visnick, entitled “Antioxidant Inflammation Modulators:Novel Derivatives of Oleanolic Acid,” filed Apr. 20, 2009; U.S. patentapplication by Xin Jiang, Xiaofeng Liu, Jack Greiner, Stephen S. Szucs,Melean Visnick entitled, “Antioxidant Inflammation Modulators: C-17Homologated Oleanolic Acid Derivatives,” filed Apr. 20, 2009.

Aqueous Solubility Determination. The following procedure was used toobtain the aqueous solubility results summarized in Example 8. Step 1.Determination of optimal UV/vis wavelengths and generation of standardcurves for a compound of interest:

-   -   (1) For eight standard calibration curves (one plate), prepare        34 mL of 50:50 (v:v) universal buffer:acetonitrile in a 50 mL        tube.    -   (2) Using a multichannel pipet, dispense (in μL) the        buffer:acetonitrile in a deep well plate as follows:

1 2 3 4 5 6 7 8 9 10 11 12 A 285 285 380 380 285 285 285 285 285 285 285285 B C D E F G H

-   -   (3) Using a multichannel pipet, dispense DMSO into the same        plate as follows:

1 2 3 4 5 6 7 8 9 10 11 12 A 12 μL 12 μL 15 μL 15 μL 15 μL 15 μL 15 μL15 μL 15 μL 15 μL B C D E F G H

-   -   (4) Add 10 mM compound in DMSO into the plates as follows:

1 2 3 4 5 6 7 8 9 10 11 12 A 15 μL 15 μL 8 μL 8 μL cmpd1 cmpd1 cmpd1cmpd1 B 15 μL 15 μL 8 μL 8 μL cmpd2 cmpd2 cmpd2 cmpd2 C 15 μL 15 μL 8 μL8 μL cmpd3 cmpd3 cmpd3 cmpd3 D 15 μL 15 μL 8 μL 8 μL cmpd4 cmpd4 cmpd4cmpd4 E 15 μL 15 μL 8 μL 8 μL cmpd5 cmpd5 cmpd5 cmpd5 F 15 μL 15 μL 8 μL8 μL cmpd6 cmpd6 cmpd6 cmpd6 G 15 μL 15 μL 8 μL 8 μL cmpd7 cmpd7 cmpd7cmpd7 H 15 μL 15 μL 8 μL 8 μL cmpd8 cmpd8 cmpd8 cmpd8

-   -   (5) Mix columns 1 and 2 by pipetting each up and down 10 times.        Mix columns 3 and 4 by pipetting up and down 10 times. Serially        dilute as follows (pipet up and down 10 times after each        transfer):

Note columns 11 and 12 contain DMSO only and so compound should not betransferred to these wells.

-   -   (6) Cover plate with lid and shake (200-300 rpm) at room        temperature for 20 minutes.    -   (7) Mix all wells by pipetting up and down 10 times.    -   (8) Transfer 120 μL from each well to a UV transparent plate.        Cover and shake for 3-5 minutes. Remove any bubbles in the wells        using a pipet.    -   (9) Read from 220 nm to 500 nm at 10 nm increments on a        spectrophotometer (e.g., SpectraMax®).

Step 2. Compound Solubility Testing Procedures using the Millipore™Multiscreen® Solubility Filter Plate.

Consumables: Millipore™ Multiscreen® Solubility Filter Plate #MSSLBPC10

-   -   Greiner® 96 well disposable UV-Star analysis plate, VWR#655801    -   Greiner® 96 well polypropylene V-bottom collection plate,        VWR#651201

Universal Aqueous Buffer:

-   -   (a) To prepare 500 mL of universal buffer, add the following:        250 mL Nanopure water; 1.36 mL (45 mM) ethanolamine; 3.08 g (45        mM) potassium dihydrogen phosphate; 2.21 g (45 mM) potassium        acetate; thoroughly mix.    -   (b) Adjust pH to 7.4 with HCl and q.s. to 500 mL with 0.15 M        KCl.    -   (c) Filter to remove particulates and reduce bacterial growth.    -   (d) Store at 4° C. in the dark.

Solubility Protocol:

-   -   (a) Add 285 μL of Universal Aqueous Buffer to desired wells of        the Millipore™ Multiscreen® Solubility filter plate.    -   (b) Add 15 μL of 10 mM compound in DMSO to the appropriate        wells. Add 15 μL of 100% DMSO only to 6 wells of the filter        plate for blanks    -   (c) Using a multichannel pipet, mix wells by pipetting up and        down 10 times. Be careful not to touch the filters in the plate        with the tips.    -   (d) Cover and gently shake (200-300 rpm) filter plate for 90        minutes at room temperature.    -   (e) Vacuum filter the aqueous solution from the Multiscreen®        solubility filter plate into a polypropylene V-bottom plate.    -   (f) Transfer 60 μL of filtrate to a UV transparent plate        (Greiner® UV-Star Analysis Plate).    -   (g) Add 60 μL of acetonitrile to each well and mix by pipetting        up and down 10 times.    -   (h) Cover and gently shake for 3-5 minutes. Remove any bubbles        with a pipet.    -   (i) Measure the absorbance of each well in the plate on the        spectrophotometer (UV/vis) at the desired wavelength. For        compounds in a plate with different absorbance peaks, set the        spectrophotometer to read a spectrum (e.g., from 220 nm to 460        nm).    -   (j) Identify concentration using measured absorbance for each        compound and the predetermined standard curve (see Step 1).

Example 2 Synthesis of Oleanolic Acid Derivatives

The synthesis of compounds 402-02 and 402-51 started from compound 1(Scheme 1). Compound 1 was oxidized with bleach to give ketone 2 in 80%yield. Formylation of 2 with ethyl formate using sodium methoxide as thebase afforded compound 402-48 (70% yield), which was then treated withhydroxylamine hydrochloride in aqueous EtOH at 55° C. to give isoxazole402-49 in 93% yield. Cleavage of the isoxazole under basic conditionsgave α-cyanoketone 402-46 in quantitative yield as a mixture of ketoneand enol forms. Compound 402-46 was treated with1,3-dibromo-5,5-dimethylhydantoin, followed by elimination of HBr usingpyridine as the base, to give compound 402-02 in 81% yield (from402-49), which was demethylated with LiI in refluxing DMF to give acid402-51 in 95% yield.

Reagents and conditions applicable to Scheme 1 are: (a) AcOH, bleach,room temperature, 1 h, 80%; (b) HCO₂Et, NaOMe, 55° C., 24 h, 70%; (c)NH₂OH.HCl, 55° C., 16 h, 93%; (d) NaOMe, 55° C., 2 h, 100%; (e) (i)1,3-dibromo-5,5-dimethylhydantoin, room temperature, 2 h; (ii) pyridine,55° C., 15 h, 81%; (f) LiI, 160° C., 8 h, 95%.

Compound 402-63 was oxidized with Dess-Martin periodinane to givealdehyde 402-64 in 47% yield (Scheme 2).

Reagents and conditions applicable to Scheme 2 are: (a) Dess-Martinperiodinane, NaHCO₃, room temperature, 1 h, 47%.

The synthesis of compounds 402-59 and 402-57 began from 402-51. Compound402-51 was treated with oxalyl chloride and catalytic DMF to give acidchloride 3. Compound 3 was treated with ammonia in methanol to give402-59 (99% from 402-51). Dehydration of 402-59 utilizing TFAA and Et₃Ngave dicyano compound 402-57 (45% yield).

Reagents and conditions applicable to Scheme 3 are: (a) (COCl)₂, DMF(cat.), 0° C. to room temperature, 3 h; (b) NH₃ (MeOH), 0° C. to roomtemperature, 5 h, 99%; (c) TFAA, Et₃N, 0° C., 3 h, 45%.

404-02 was synthesized from compound 3 as summarized in Scheme 4.Compound 3 was reacted with 2,2,2-trifluoroethylamine-HCl in toluene andwater at 70° C. with NaHCO₃ as the base, giving 404-02 in 69% yield.

Reagents and conditions applicable to Scheme 4 are: (a)2,2,2-trifluoroethylamine-HCl, NaHCO₃, toluene, H₂O, 70° C., 69%.

Reagents and conditions applicable to Scheme 5 are: (a) LiAlH₄, THF, 0°C., 1.5 h, 38% for 4; 34% for 5; (b) NaOMe, 55° C., 7 h, 94% for 6; 89%for 7; (c) (i) 1,3-dibromo-5,5-dimethylhydantoin, r.t., 40 min; (ii)pyridine, 55° C., 25 h, 35% for 402-66; 32% for 63219.

The synthesis of 402-66 began from isoxazole compound 402-49. Reductionof the ketone in 402-49 was achieved by treatment with LiAlH₄ in THF at0° C., giving compounds 4 and 5 (as a 1:1 mixture of diastereotopicalcohols). Compound 4 was treated with NaOMe in MeOH at 55° C. to give6, which exists as a 3:2 mixture of keto and enol tautomeric forms.Bromination and subsequent dehydrobromination of 6 by treatment with1,3-dibromo-5,5-dimethylhydantoin and then pyridine gave 402-66 in 33%yield (from 4). Using the same synthetic sequence, compound 5 wasconverted into 63219 in 28% overall yield.

Reagent and conditions applicable to Scheme 6 are: (a) oxalyl chloride,r.t., 2 h; (b) NH₂NH₂—H₂O, 0° C., 30 min, 97%; (c) AcCl, Et₃N, r.t., 1h, 77%; (d) NaOMe, r.t., 10 min, 72%; (e) TsOH, 110° C., 1 h, 33%.

Reagent and conditions applicable to Scheme 7 are: (a) Et₃N, TFAA, r.t.,1.5 h, 85%; (b) Bu₃SnN₃, 150° C., 67%; (c) (i) HCO₂Et, NaOMe, 0° C. toroom temperature, 1 h; (ii) NH₂OH.HCl, 60° C., 3 h, 54%; (d) NaOMe, 55°C., 2 h; (e) 1,3-dibromo-5,5-dimethylhydantoin, r.t., 2 h; (f) pyridine,55° C., 16 h, 60% for 63229; 69% for 63230; (g) TMSCHN₂, 0° C., 10 min,77%.

Reagents and conditions applicable to Scheme 8 are: (a) i) NaOMe,HCO₂Et, 0° C. to room temperature; concentrated HCl ii) NH₂OH—HCl, EtOH,H₂O, 60° C., 14 h, 47%; (b) NaOMe, 55° C., 16 h, quantitative; (c) (i)1,3-dibromo-5,5-dimethylhydantoin, room temperature, 3 h; (ii) pyridine,55° C., 16 h, 12%.

Reagents and conditions applicable to Scheme 9 are: (a) LiAlH₄, THF, 0°C., 2 h, 19%; (b) NaOMe, 55° C., 7 h, 92%; (c) (i)1,3-dibromo-5,5-dimethylhydantoin, r.t., 50 min; (ii) pyridine, 55° C.,7 h, 56%.

Reagents and conditions pertaining to Scheme 10: (a) (i) HCO₂Et, NaOMe,0° C., 1.5 h; (ii) NH₂OH—HCl, 65° C., 3.5 h, 78%; (b) (i) oxalylchloride, 0° C. to rt, 2 h; (ii) AcNHNH₂, Et₃N, 0° C. to rt, 30 min,99%; (c) Lawesson's reagent, 110° C., 30 min, 10% (for compound 21) and29% (for compound 22); (d) NaOMe, 55° C., 2 h, 73%; (e) (i) DBDMH, 0°C., 1 h; (ii) pyridine, 55° C., 3 h, 79%. Compound 18 was reported byHonda et al. (2000a), which is incorporated herein by reference in itsentirety.

Reagents and conditions applicable to Scheme 11: (a) LAH, rt to 65° C.,1.5 h, 27%; (b) TEMPO, IPh(OAc)₂, rt, 72 h, 77%; (c)(Ph₃PCH₂Cl)Cl/n-BuLi, THF, HMPA, 0° C. to rt, 87%; (d) MeLi, THF, 0° C.to rt, 91%; (e) PCC, NaOAc, CH₂Cl₂, rt, 78%; (f) HCO₂Et, NaOMe, 0° C. tort; (g) NH₂OHHCl, EtOH—H₂O, 60° C., 89% from 28; (h) NaOMe, 55° C., 3 h;(i) DDQ, Benzene, 85° C., 39% from 30.

Reagents and conditions applicable to Scheme 12 are: (a) HgSO₄, H₂SO₄,acetone/H₂O, 55° C., 20 h, 91%; (b) (i)1,3-dibromo-5,5-dimethylhydantoin, 0° C., 3 h; (ii) pyridine, 55° C., 19h, 73%.

Reagents and conditions pertaining to Scheme 13: (a) benzyl bromide,DBU, 100° C., 6 h, 65%.

Reagents and conditions pertaining to Scheme 14: (a) MeONH₂—HCl, Et₃N,40° C., 4 h, 37%.

Reagents and conditions pertaining to Scheme 15: (a) Me₂NH, 40° C., 71h, 61%.

Reagents and conditions applicable to Scheme 16 are: (a) NH₂OH—HCl,NaOAc, EtOH, H₂O, 80° C., 27 h, 72%; (b) NaOMe, MeOH, 55° C., 1 h; (c)(i) 1,3-dibromo-5,5-dimethylhydantoin, 0° C., 40 min; (ii) pyridine, 55°C., 7 h, 27% from 33.

Reagents and conditions applicable to Scheme 17 are: (a) POCl₃,pyridine, rt, 5 h, 75%; (b) NaOMe, MeOH, 55° C., 4 h, 93% (i)1,3-dibromo-5,5-dimethylhydantoin, 0° C., 1 h; (ii) pyridine, 55° C., 23h, 93%.

Reagents and conditions applicable to Scheme 18 are: (a) m-CPBA, rt, 48h, 22%; (b) NaOMe, 55° C., 2 h, 66%; (c) (i) DBDMH, 0° C., 1 h; (ii)pyridine, 55° C., 3 h, 72%.

Reagents and conditions pertaining applicable to Scheme 19 are: (a)LiAlH₄, 0° C., 40 min, 62%; (b) NaOMe, 55° C., 2 h, 83%; (c) (i) DBDMH,0° C., 1 h; (ii) pyridine, 55° C., 4 h, 40%.

Example 3 Synthesis and Characterization of Oleanolic Acid Derivatives

Compound 2: Bleach (5.25 wt % of NaClO (aq), 129 mL, 91 mmol) was addedto a stirred solution of compound 1 (34.67 g, 71 mmol) in AcOH (471 mL)at room temperature. After stirring for 40 min, the reaction mixture waspoured into ice-water (1.5 L) and stirred for 5 min. The whiteprecipitate was collected by filtration and washed thoroughly withwater. The filtered solid was then dissolved in EtOAc and washed withNaHCO₃ (aq) solution, dried with MgSO₄ and concentrated. The residueobtained was purified by column chromatography (silica gel, 10% to 25%EtOAc in hexanes) to give product 2 (27.8 g, 80%) as a white foam solid:¹H NMR (400 MHz, CDCl₃) δ 3.69 (s, 3H), 2.80 (m, 1H), 2.65 (d, 1H, J=4.0Hz), 2.53 (ddd, 1H, J=7.2, 10.8, 16.0 Hz), 2.38 (ddd, 1H, J=3.6, 6.8,16.0 Hz), 2.16-2.30 (m, 2H), 1.95 (m, 1H), 1.89 (m, 1H), 1.80 (m, 2H),1.62-1.73 (m, 3H), 1.57 (m, 2H), 1.47 (m, 2H), 1.15-1.40 (m, 7H), 1.09(s, 3H), 1.05 (s, 3H), 1.01 (s, 3H), 0.99 (s, 3H), 0.98 (s, 3H), 0.95(s, 3H), 0.90 (s, 3H); m/z 485.3 (M+1).

Compound 402-48: NaOMe solution (25% w/w in MeOH, 132.3 mL, 570 mmol)was added to a solution of compound 2 (27.6 g, 57 mmol) in MeOH (250 mL)under nitrogen. The reaction mixture was heated to 55° C. in an oilbath, and HCO₂Et (93 mL, 1.15 mmol, 20 eq) was added dropwise via anaddition funnel. The reaction mixture was stirred at 55° C. for 24 h andthen at room temperature for another 40 h. After removal of MeOH (150mL) by evaporation, t-BuOMe (200 mL) was added, and the mixture wascooled to 0° C. 12 N HCl (aq) (50 mL, 600 mmol, 10.5 eq) was then addedover 10 min, and the mixture was extracted with EtOAc. The combinedextracts were washed with water, dried with MgSO₄, and concentrated. Thebrown oil obtained was purified by column chromatography (silica gel, 5%to 10% EtOAc in hexanes) to give product 402-48 (20.5 g, 70%) as a whitefoam solid: ¹H NMR (400 MHz, CDCl₃) δ 14.89 (d, 1H, J=3.2 Hz), 8.61 (d,1H, J=3.2 Hz), 3.69 (s, 3H), 2.80 (m, 1H), 2.67 (d, 1H, J=4.0 Hz),2.20-2.34 (m, 3H), 1.98 (m, 1H), 1.62-1.92 (m, 6H), 1.10-1.56 (m, 10H),1.20 (s, 3H), 1.12 (s, 3H), 1.02 (s, 3H), 0.99 (s, 3H), 0.96 (s, 3H),0.91 (s, 3H), 0.85 (s, 3H); m/z 513.3 (M+1).

Compound 402-49: A mixture of compound 402-48 (20.3 g, 40 mmol) andNH₂OH.HCl (4.12 g, 59 mmol) in EtOH (300 mL) and water (60 mL) washeated at 55° C. for 14 h. After cooling to room temperature, EtOH wasremoved by evaporation, and the white slurry obtained was extracted withEtOAc. The combined extracts were washed with water, dried with MgSO₄,and concentrated. The residue obtained was purified by columnchromatography (silica gel, 10% to 20% EtOAc in hexanes) to give product402-49 (18.8 g, 93%) as a white solid: ¹H NMR (400 MHz, CDCl₃) δ 7.99(s, 1H), 3.70 (s, 3H), 2.81 (m, 1H), 2.68 (d, 1H, J=4.4 Hz), 2.37 (d,1H, J=15.2 Hz), 2.23-2.33 (m, 2H), 1.76-1.98 (m, 5H), 1.68 (m, 3H),1.11-1.62 (m, 9H), 1.32 (s, 3H), 1.23 (s, 3H), 1.02 (s, 3H), 0.99 (s,3H), 0.97 (s, 3H), 0.91 (s, 3H), 0.84 (s, 3H); m/z 510.3 (M+1).

Compound 402-46: NaOMe (25% w/w in MeOH, 8.75 mL, 38 mmol) was addeddropwise to a suspension of 402-49 (16.16 g, 31.7 mmol) in MeOH (55 mL)at 0° C. under N₂. The reaction mixture was heated at 55° C. for 2 h andthen cooled to 0° C. t-BuOMe (150 mL) and 1 N HCl (aq) (50 mL) wereadded successively, and the mixture was extracted with EtOAc. Thecombined extracts were washed with water, dried with MgSO₄, andconcentrated to give compound 402-46 (17.80 g, 100%) as a white foamsolid. 402-46 is a mixture of two equilibrium forms, the enol form (asshown in Scheme 1) and the ketone form, in the ratio of 2:3. ¹H NMR ofthe mixture: (400 MHz, CDCl₃) δ 5.69 (s, 0.4H), 3.87 (m, 0.6H), 2.80 (m,1H), 2.65 (m, 1H), 0.82-2.30 (m, 44H); m/z 510.3 (M+1).

Compound 402-02: 1,3-Dibromo-5,5-dimethylhydantoin (5.98 g, 20.9 mmol)was added to a solution of compound 402-46 (17.76 g, 35 mmol) in DMF (75mL) at 10° C. After stirring at room temperature for 2 h, pyridine (8.5mL, 105 mmol) was added, and the reaction mixture was heated at 55° C.for 15 h. After cooling to room temperature, the mixture was poured intowater (700 mL) and stirred for 5 min. The pale brown precipitate wascollected by filtration and washed with water. The solid was dissolvedin CH₂Cl₂, and the solution was washed with 1 N HCl (aq) and water, thendried with MgSO₄ and concentrated. The residue obtained was purified bycolumn chromatography (silica gel, 0% to 70% EtOAc in hexanes) to giveproduct 402-02 (14.3 g, 81%) as a white solid: ¹H NMR (400 MHz, CDCl₃) δ7.65 (s, 1H), 3.69 (s, 3H), 2.82 (m, 1H), 2.68 (d, 1H, J=4.4 Hz), 2.44(dd, 1H, J=4.8, 16.0 Hz), 2.35 (dd, 1H, J=12.8, 16.0 Hz), 1.86-2.00 (m,3H), 1.81 (m, 1H), 1.60-1.71 (m, 4H), 1.42-1.55 (m, 3H), 1.24 (m, 1H),1.10-1.24 (m, 4H), 1.22 (s, 3H), 1.16 (s, 3H), 1.15 (s, 3H), 1.07 (s,3H), 0.99 (s, 3H), 0.97 (s, 3H), 0.92 (s, 3H); m/z 508.2 (M+1).

Compound 402-51: A stream of nitrogen was bubbled through a stirringsolution of compound 402-02 (6.31 g, 12.4 mmol) and LiI (33.35 g, 248mmol) in DMF (87 mL) at 160° C. for 8 h. After cooling to 50° C., thereaction mixture was diluted with EtOAc (100 mL). 1 N HCl (aq) solution(30 mL) was then added at room temperature and stirred for 5 min. Themixture was extracted with EtOAc, and the combined extracts were washedwith water, 10% Na₂S₂O₃ (aq) and water, then dried with MgSO₄ andconcentrated. The residue obtained was purified by column chromatography(silica gel, 5% to 50% EtOAc in CH₂Cl₂) to give acid 402-51 (6.02 g,95%) as a white solid: ¹H NMR (400 MHz, CDCl₃) δ 10.41 (bs, 1H), 7.65(s, 1H), 2.80 (m, 1H), 2.74 (d, 1H, J=4.4 Hz), 2.46 (dd, 1H, J=4.8, 16.0Hz), 2.37 (dd, 1H, J=12.8, 16.0 Hz), 1.86-2.02 (m, 4H), 1.44-1.79 (m,8H), 1.35 (m, 1H), 1.12-1.29 (m, 3H), 1.22 (s, 3H), 1.16 (s, 3H), 1.14(s, 3H), 1.11 (s, 3H), 0.99 (s, 3H), 0.98 (s, 3H), 0.93 (s, 3H); m/z494.3 (M+1).

Compound 402-64: NaHCO₃ (78 mg, 0.93 mmol) and Dess-Martin periodinane(99 mg, 0.23 mmol) were added successively to a solution of 402-63 (45mg, 94 μmol) in CH₂Cl₂ (5 mL) at room temperature. After stirring for 1h, 5% Na₂S₂O₃ (aq) solution was added. The reaction mixture wasextracted with t-BuOMe, and the combined extracts were washed withNaHCO₃ (aq) solution, dried with MgSO₄, and concentrated. The crudeproduct obtained was purified by column chromatography (silica gel, 0%to 35% EtOAc in hexanes) to give 402-64 (22 mg, 49%) as a white foamsolid: ¹H NMR (300 MHz, CDCl₃) δ 9.33 (s, 1H), 7.62 (s, 1H), 2.61 (m,1H), 2.50 (d, 1H, J=4.4 Hz), 2.45 (dd, 1H, J=4.8, 16.4 Hz), 2.34 (dd,1H, J=13.2, 16.4 Hz), 1.92-2.00 (m, 2H), 1.88 (m, 1H), 1.42-1.74 (m,9H), 1.28-1.35 (m, 2H), 1.21 (s, 3H), 1.20 (m, 1H), 1.15 (s, 3H), 1.14(s, 3H), 1.12 (m, 1H), 1.06 (s, 3H), 0.97 (s, 6H), 0.93 (s, 3H); m/z478.2 (M+1).

Compound 402-59: To a solution of 402-51 (2.08 g, 4.21 mmol) in CH₂Cl₂(28 mL) were successively added oxalyl chloride (1.07 mL, 12.64 mmol)and DMF (5 drops, cat.) at 0° C. The reaction was allowed to warm toroom temperature and was stirred for 3 h. The reaction mixture wasconcentrated and dried in vacuo 30 min, to give acid chloride 3 as ayellow solid, which was used directly in the next step. To a solution of3 (2.16 g, 4.21 mmol) in THF (28 mL) at 0° C. was added ammonia (2.0 Msolution in MeOH, 11 mL, 22.00 mmol). The reaction was allowed to warmto room temperature and was stirred for 5 h. The solvents were thenevaporated, and the residue was extracted with EtOAc. The extracts werewashed with water, 1 N HCl (aq), and water, then dried over MgSO₄,filtered, and concentrated to give 402-59 (2.06 g, 99%) as a pale yellowsolid. A small amount (53 mg) was purified by column chromatography(silica gel, 0% to 25% EtOAc in CH₂Cl₂) to give higher purity 402-59 (14mg, white solid) for biological assay: ¹H NMR (400 MHz, CDCl₃) δ 7.65(s, 1H), 5.63 (br s, 1H), 5.36 (br s, 1H), 2.90 (br d, 1H, J=5.2 Hz),2.71 (br d, 1H, J=12 Hz), 2.42 (m, 2H), 1.96-2.10 (m, 4H), 1.78-1.90 (m,2H), 1.45-1.69 (m, 6H), 1.23-1.40 (m, 4H), 1.22 (s, 3H), 1.16 (s, 3H),1.15 (s, 3H), 1.13 (s, 3H), 0.99 (s, 3H), 0.98 (s, 3H), 0.93 (s, 3H);m/z 493.3 (M+1)

Compound 402-57: A solution of 402-59 (2.01 g, 4.08 mmol) in CH₂Cl₂ (28mL) was prepared and cooled to 0° C. To this solution were added TFAA(0.91 mL, 6.55 mmol) and Et₃N (1.48 mL, 10.62 mmol). The reaction wasstirred at 0° C. for 3 h, after which it was quenched by the addition ofsaturated NaHCO₃ (aq) solution (40 mL). After stirring for 10 min, thereaction mixture was extracted with CH₂Cl₂ and washed with saturatedNaHCO₃ (aq), water, 1 N HCl (aq), and water. The extracts were driedover MgSO₄, filtered, and concentrated. The crude product was purifiedby column chromatography (silica gel, 5% to 35% EtOAc in hexanes). Thepurified product was triturated with EtOH, then filtered and dried onthe filter to give 402-57 (0.87 g, 45%) as a powdery white solid: ¹H NMR(400 MHz, CDCl₃) δ 7.63 (s, 1H), 3.04 (d, 1H, J=4.4 Hz), 2.38-2.57 (m,3H), 1.91-2.19 (m, 5H), 1.61-1.78 (m, 4H), 1.44-1.54 (m, 3H), 1.32 (s,3H), 1.26-1.30 (m, 4H), 1.22 (s, 3H), 1.19 (s, 3H), 1.16 (s, 3H), 0.99(s, 3H), 0.95 (s, 3H), 0.92 (s, 3H); m/z 475.2 (M+1).

Compound 404-O₂: To a solution of 3 (3.03 g, 5.92 mmol) in toluene (84mL) was added NaHCO₃ (1.98 g). A solution of trifluoroethylaminehydrochloride (5.64 g, 41.62 mmol) in water (14 mL) was prepared, thenadded to the reaction. The reaction was heated to 70° C. and stirred for2 h. After cooling to room temperature, the reaction mixture wasextracted with EtOAc and washed with brine. The combined extracts weredried over MgSO₄, filtered, and concentrated. The crude product waspurified by column chromatography (silica gel, 0% to 40% EtOAc inCH₂Cl₂) to give 404-02 (2.35 g, 69%) as a white solid: ¹H NMR (400 MHz,CDCl₃) δ 7.65 (s, 1H), 5.94 (br t, 1H, J=8 Hz), 4.10 (m, 1H), 3.69-3.88(m, 1H), 2.84 (d, 1H, J=8 Hz), 2.78 (br d, 1H, J=16 Hz), 2.38 (m, 2H),2.12 (m, 1H), 2.06 (m, 2H), 1.61-1.83 (m, 5H), 1.24-1.52 (m, 8H), 1.22(s, 3H), 1.16 (s, 3H), 1.14 (s, 3H), 1.07 (s, 3H), 0.99 (s, 6H), 0.93(s, 3H); m/z 575.3 (M+1).

Compounds 4, 5: To a solution of 402-49 (395 mg, 0.775 mmol) in THF (7.8mL) was added LiAlH₄ (1.0 M solution in THF, 0.78 mL, 0.780 mmol) at 0°C. The reaction was stirred at 0° C. for 40 min, after which it wasquenched by the addition of water (5 mL) and stirred 5 min. The reactionmixture was extracted with EtOAc and washed with water. Solid NaCl wasadded to break up emulsions. The combined extracts were dried overNa₂SO₄, filtered, and concentrated. The crude product was purified bycolumn chromatography (silica gel, 10% to 70% EtOAc in hexanes) to giveboth 4 (151 mg, 38%) as a white solid and to give 5 (134 mg, 34%) as awhite solid: Compound 4: ¹H NMR (400 MHz, CDCl₃) δ 7.99 (s, 1H), 3.79(m, 1H), 3.72 (s, 3H), 2.75 (m, 1H), 2.52 (d, 1H, J=14.4 Hz), 1.95-2.11(m, 2H), 1.57-1.88 (m, yyH), 1.24-1.54 (m, yyH), 1.30 (s, 3H), 1.20 (s,3H), 1.00 (s, 3H), 0.93 (s, 6H), 0.92 (s, 3H), 0.82 (s, 3H); m/z 512.3(M+1).

Compound 5: m/z 494.3 (M−17), 434.3 (M−17−60).

Compound 6: To a solution of 4 (371 mg, 66 μmol) in MeOH (7.3 mL) wasadded NaOMe (25 wt % solution in MeOH, 0.42 mL, 1.837 mmol) at roomtemperature. The reaction was heated to 55° C. and stirred 7 h. Aftercooling the reaction to room temperature, the reaction mixture wasdiluted with MTBE (10 mL), then quenched with 1 N HCl (aq) (10 mL). Thereaction mixture was extracted with EtOAc and washed with 1 N HCl (aq)and brine. The combined extracts were dried over Na₂SO₄, filtered, andconcentrated to give 6 (361 mg, 94%) as a white solid. Compound 6 is amixture of two equilibrium forms, the enol form (as shown in Scheme 5)and the ketone form, in the ratio of 2:3. ¹H NMR of the mixture: (400MHz, CDCl₃) δ 5.66 (d, 0.4H, J=4.8 Hz), 4.09 (br, 1H), 3.90 (m, 0.6H),3.71 (s, 1.2H), 3.68 (s, 1.8H), 2.73 (m, 1H), 2.48 (m, 1H), 2.14-2.26(m, 2H), 0.80-2.02 (m, 39H); m/z 494.3 (M−17), 434.3 (M−77).

Compound 402-66: A solution of 6 (361 mg, 0.705 mmol) in DMF (7.1 mL)was prepared. 1,3-Dibromo-5,5-dimethylhydantoin (120 mg, 0.420 mmol) wasadded, and the reaction was stirred at room temperature for 1 h.Pyridine (0.23 mL, 2.858 mmol) was added, and the reaction was heated to55° C. and stirred 10 h. After cooling the reaction to room temperature,the reaction mixture was extracted with EtOAc and washed with 5% Na₂S₂O₃(aq), water, 1 N HCl (aq), and water. The EtOAc extracts were dried overNa₂SO₄, filtered, and evaporated. The crude product was purified bycolumn chromatography (silica gel, 5% to 40% EtOAc in hexanes) to give402-66 (127 mg, 35% yield) as a white solid; ¹H NMR (400 MHz, CDCl₃) δ7.81 (s, 1H), 3.81 (ddd, 1H, J=4.8, 10.8, 15.6 Hz), 3.72 (s, 3H), 2.75(m, 1H), 2.01 (m, 2H), 1.74-1.88 (m, 3H), 1.24-1.72 (m, 15H), 1.19 (s,3H), 1.13 (s, 3H), 1.12 (s, 3H), 0.98 (s, 3H), 0.96 (s, 3H), 0.95 (s,3H), 0.94 (s, 3H); m/z 492.3 (M−17), 432.3 (M−77).

Compound 7: Using the procedure described for the synthesis of compound6 from compound 4, compound 7 (96 mg, 89% yield) was produced fromcompound 5 (108 mg, 0.211 mmol): m/z 494.3 (M−17).

Compound 63219: Using the procedure described for the synthesis ofcompound 402-66 from compound 6, compound 63219 (30 mg, 32% yield) wasproduced from compound 7 (95 mg, 0.186 mmol): ¹H NMR (400 MHz, CDCl₃) δ7.81 (1H, s), 4.16 (1H, bs), 3.68 (3H, s), 2.44-2.54 (2H, m), 1.98-2.10(2H, m), 1.78-1.94 (4H, m), 1.42-1.76 (7H, m), 1.00-1.42 (6H, m), 1.32(3H, s), 1.23 (3H, s), 1.13 (3H, s), 1.11 (3H, s), 0.94 (3H, s), 0.93(3H, s), 0.92 (3H, s); m/z 492.3 (M−17).

Compound 8: Oxalyl chloride (0.11 mL, 1.30 mmol) and catalytic amount ofDMF were added sequentially to a solution of compound 402-51 (200 mg,0.41 mmol) in CH₂Cl₂ (4 mL) at 0° C. The reaction mixture was warmed toroom temperature and stirred for 2 h. After removing the solvent byevaporation, the crude acid chloride was obtained as a light yellow foamsolid. Hydrazine hydrate (64% of hydrazine, 0.50 mL) was added to asolution of acid chloride in Et₂O (8 mL) at 0° C. After stirring for 30min, CH₂Cl₂ was added. The mixture was washed with water, dried overMgSO₄, filtered and evaporated to give compound 8 (200 mg, 97% yield) aswhite solid, which was used in the next step without furtherpurification: m/z 508.3 (M+1).

Compound 9: Et₃N (0.12 mL, 0.86 mmol) and acetyl chloride (37 μL, 0.52mmol) were added sequentially to a solution of compound 8 (200 mg, 0.39mmol) in CH₂Cl₂ (4 mL) at r.t. After stirring for 30 min, Et₃N (0.36 mL,2.59 mmol) and acetyl chloride (110 μL, 1.55 mmol) were added again.After stirring for another 30 min, NaHCO₃ (aq.) solution was added toquench the reaction. The reaction mixture was transferred to aseparatory funnel, and extracted with EtOAc. The combined extracts werewashed with water, dried over MgSO₄, filtered and evaporated. Theresidue was purified by silica gel chromatography (0% to 75% EtOAc inhexanes) to give compound 9 (180 mg, 77% yield) as a white foam solid:m/z 592.3 (M+1).

Compound 63264: NaOMe (25% w/w in MeOH, 0.14 mL, 0.61 mmol) was added toa solution of compound 9 (180 mg, 0.30 mmol) in MeOH (3 mL) at 0° C.After stirring at r.t. for 10 min, the reaction mixture was treated witht-BuOMe (10 mL) and 1N HCl (aq.) (1 mL), which was then transferred to aseparatory funnel and extracted with EtOAc. The combined extracts werewashed with NaHCO₃ (aq.) solution, dried over MgSO₄, filtered andevaporated. The residue was purified by silica gel chromatography (0% to100% EtOAc in hexanes) to give compound 63264 (121 mg, 72% yield) as awhite solid: ¹H NMR (400 MHz, CDCl₃) δ 7.98 (d, 1H, J=4.4 Hz), 7.77 (d,1H, J=4.4 Hz), 7.65 (s, 1H), 2.89 (d, 1H, J=4.4 Hz), 2.82 (m, 1H),2.34-2.45 (m, 2H), 2.10 (m, 1H), 2.08 (s, 3H), 1.82-2.02 (m, 4H),1.60-1.69 (m, 3H), 1.44-1.53 (m, 4H), 1.16-1.40 (m, 4H), 1.22 (s, 3H),1.16 (s, 3H), 1.15 (s, 3H), 1.13 (s, 3H), 0.99 (s, 3H), 0.98 (s, 3H),0.94 (s, 3H); m/z 550.3 (M+1).

Compound 63267: A solution of compound 63264 (74 mg, 0.13 mmol), TsOH(13 mg, 0.068 mmol) in toluene (5 mL) was heated at reflux withdean-stark trap for 1 h. After cooling to r.t., the reaction mixture wastransferred to a separatory funnel, washed with NaHCO₃ (aq.) solution,dried over MgSO₄, filtered and evaporated. The residue was purified bysilica gel chromatography (0% to 100% EtOAc in hexanes) to give compound63267 (24 mg, 33% yield) as a white foam solid: ¹H NMR (400 MHz, CDCl₃)δ 7.64 (s, 1H), 2.94 (m, 1H), 2.79 (d, 1H, J=4.4 Hz), 2.54 (s, 3H), 2.46(m, 1H), 2.34 (m, 1H), 2.21 (m, 1H), 1.84-2.06 (m, 5H), 1.56-1.70 (m,4H), 1.24-1.47 (m, 5H), 1.23 (s, 3H), 1.18 (m, 1H), 1.15 (s, 3H), 1.13(s, 3H), 1.05 (s, 3H), 1.02 (s, 3H), 0.98 (s, 3H), 0.93 (s, 3H); m/z532.3 (M+1).

Compound 11: Et₃N (1.46 mL, 10.49 mmol) and TFAA (0.88 mL, 6.33 mmol)were added sequentially to a solution of compound 10 (1.97 g, 4.20 mmol)in CH₂Cl₂ (42 mL) at 0° C. After stirring for 1.5 h, NaHCO₃ (aq.)solution was added to the reaction mixture, which was then transferredto a separatory funnel and extracted with CH₂Cl₂. The combined extractswere dried over MgSO₄, filtered and evaporated. The residue was purifiedby silica gel chromatography (0% to 35% EtOAc in hexanes) to givecompound 11 (1.62 g, 85% yield) as a white solid: m/z 452.3.

Compound 12: A solution of Bu₃SnN₃ (1.00 mL, 3.62 mmol) and compound 11(1.36 g, 3.02 mmol) in xylene (5.0 mL) was heated at reflux for 48 h.After cooling to r.t., the reaction mixture was purified by silica gelchromatography (0% to 30% EtOAc in CH₂Cl₂) to give compound 12 (994 mg,67% yield) as a light yellow foam solid: m/z 493.3 (M+1).

Compound 13: NaOMe solution (25% w/w in MeOH, 1.16 mL, 5.07 mmol) wasadded dropwise to a mixture of compound 12 (168 mg, 0.34 mmol) andHCO₂Et (0.82 mL, 10.19 mmol) at 0° C. under N₂. After stirring at roomtemperature for 1 h, t-BuOMe (10 mL) was added. The mixture was cooledto 0° C., and 12 N HCl (aq) (0.42 mL, 5.04 mmol) was added slowly. Themixture was transferred to a separatory funnel and extracted with EtOAc.The combined extracts were washed with water, dried over MgSO₄ andconcentrated to give crude 2-formyl ketone, which was then mixed withNH₂OH.HCl (36 mg, 0.51 mmol), EtOH (4 mL), and water (0.4 mL), andheated at 60° C. for 3 h. After removing EtOH by evaporation, the whiteslurry obtained was transferred to a separatory funnel and extractedwith CH₂Cl₂. The combined extracts were washed with water, dried overMgSO₄, and concentrated. The residue was purified by columnchromatography (silica gel, 0% to 30% EtOAc in CH₂Cl₂) to give compound13 (95 mg, 54% yield) as a white foam solid: m/z 520.3 (M+1).

Compound 63229: Using the procedure described for the synthesis ofcompound 402-66 from compound 4, 63229 (12 mg, 60% yield) was producedfrom compound 13 (20 mg, 0.038 mmol): ¹H NMR (400 MHz, CDCl₃) δ 7.70 (s,1H), 3.03 (m, 1H), 2.69 (d, 1H, J=4.0 Hz), 2.52 (dd, 1H, J=4.4, 16.8Hz), 2.29-2.36 (m, 2H), 1.96-2.03 (m, 3H), 1.56-1.82 (m, 6H), 1.25-1.57(m, 6H), 1.22 (s, 3H), 1.18 (m, 1H), 1.13 (s, 3H), 1.11 (s, 3H), 1.04(s, 3H), 1.03 (s, 3H), 0.98 (s, 3H), 0.75 (s, 3H); m/z 518.3 (M+1).

Compound 14: TMSCHN₂ (2.0 M in Et₂O, 89 mL, 0.18 mmol) was added to asolution of compound 13 (84 mg, 0.16 mmol) in THF (1.25 mL) and MeOH(0.31 mL) at 0° C. After stirring at room temperature for 10 min, aceticacid was added to quench the reaction. The reaction mixture was dilutedwith EtOAc, transferred to a separatory funnel, washed with NaHCO₃ (aq.)solution, dried over MgSO₄ and evaporated. The residue was purified bycolumn chromatography (silica gel, 0% to 60% EtOAc in hexanes) to givecompound 14 (67 mg, 77% yield) as a white solid: m/z 534.3 (M+1).

Compound 63230: Using the procedure described for the synthesis ofcompound 402-66 from compound 4, 63230 (45 mg, 69% yield) was producedfrom compound 14 (65 mg, 0.12 mmol): ¹H NMR (400 MHz, CDCl₃) δ 7.62 (s,1H), 4.32 (s, 3H), 3.11 (m, 1H), 2.68 (d, 1H, J=4.4 Hz), 2.42 (dd, 1H,J=4.8, 16.4 Hz), 2.27 (dd, 1H, J=13.2, 16.4 Hz), 2.22 (dd, 1H, J=4.4,14.8 Hz), 1.94-2.04 (m, 3H), 1.79 (m, 1H), 1.54-1.63 (m, 5H), 1.36-1.50(m, 4H), 1.26 (m, 1H), 1.20 (s, 3H), 1.13 (m, 1H), 1.12 (s, 3H), 1.09(s, 3H), 1.05 (s, 3H), 1.01 (s, 3H), 0.97 (s, 3H), 0.70 (s, 3H); m/z532.3 (M+1).

Compound 63223: Using the procedure described for the synthesis ofcompound 13 from compound 12, compound 63223 (1.95 g, 47% yield) wasproduced from compound 15 (3.93 g, 7.12 mmol) as a pale yellow solid: ¹HNMR (400 MHz, CDCl₃) δ 7.98 (1H, s), 5.91 (1H, t, J=6.0 Hz), 4.00-4.15(1H, m), 3.55-3.90 (1H, m), 2.72-2.82 (2H, m), 2.20-2.40 (3H, m),1.88-2.16 (4H, m), 1.10-1.84 (13H, m), 1.31 (3H, s), 1.21 (3H, s), 1.00(3H, s), 0.98 (6H, s), 0.91 (3H, s), 0.82 (3H, s); m/z 577.3 (M+H).

Compound 63227: Using the procedure described for the synthesis ofcompound 6 from compound 4, compound 63227 (1.64 g, quantitative yield)was produced from compound 63223 (1.61 g, 2.79 mmol): ¹H NMR (400 MHz,CDCl₃) for enol form: δ 5.91 (1H, t, J=6.0 Hz), 5.78 (1H, bs, enol),4.00-4.16 (1H, m), 3.75-3.94 (1H, m), 2.70-2.85 (2H, m), 1.90-2.30 (5H,m), 0.80-1.88 (36H, m); m/z 577.3 (M+H) (for both enol and ketoneisomers).

Compound 63237: A solution of 63227 (1.61 g, 2.79 mmol) in DMF (9.3 mL)was prepared. 1,3-Dibromo-5,5-dimethylhydantoin (456 mg, 1.59 mmol) wasadded, and the reaction was stirred at room temperature for 3 h.Pyridine (0.67 mL, 8.33 mmol) was added, and the reaction was heated to55° C. and stirred 16 h. After cooling the reaction to room temperature,the reaction mixture was extracted with EtOAc and washed with 5% Na₂S₂O₃(aq), 1 N HCl (aq), and water. The EtOAc extracts were dried overNa₂SO₄, filtered, and evaporated. Compound 63237 was a minor component(18%) by crude LC-MS analysis. The crude product was purified by columnchromatography (silica gel, 5% to 35% EtOAc in hexanes) to give 63237(188 mg, 12%) as a yellow foam solid: ¹H NMR (400 MHz, CDCl₃) δ 7.67 (s,1H), 3.82 (m, 2H), 2.91 (m, 1H), 2.54 (m, 1H), 2.41 (m, 1H), 2.08 (m,1H), 1.83-1.94 (m, 2H), 1.52-1.74 (m, 6H), 1.39 (s, 3H), 1.22 (s, 6H),1.20-1.49 (m, 7H), 1.16 (s, 3H), 0.98 (s, 3H), 0.96 (s, 3H), 0.95 (s,3H); m/z 573.3 (M+1).

Compound 16: Using the procedure described for the synthesis of compound4 from compound 402-49, compound 16 (100 mg, 19% yield) was producedfrom compound 63223 (531 mg, 0.921 mmol): m/z 579.3 (M+1)

Compound 17: Using the procedure described for the synthesis of compound6 from compound 4, compound 17 (90 mg, 92% yield) was produced fromcompound 16 (98 mg, 0.169 mmol): m/z 561.3 (M−17).

Compound 63268: Using the procedure described for the synthesis ofcompound 402-66, compound 63268 (50 mg, 56% yield) was produced fromcompound 17 (90 mg, 0.156 mmol): ¹H NMR (400 MHz, CDCl₃) δ 7.78 (s, 1H),6.09 (t, 1H, J=6.4 Hz), 4.06 (m, 1H), 3.89 (m, 1H), 3.78 (m, 1H), 2.70(m, 1H), 1.98-2.05 (m, 2H), 1.84 (dd, 1H, J=4.4, 10.8 Hz), 1.75 (ddd,1H, J=4.4, 13.6, 13.6 Hz), 1.20-1.62 (m, 15H), 1.18 (s, 3H), 1.11 (s,3H), 1.10 (s, 3H), 1.06 (m, 1H), 0.98 (s, 3H), 0.95 (s, 3H), 0.94 (s,3H), 0.91 (s, 3H); m/z 559.3 (M−17).

Compound 19: NaOMe (25 w/w % solution in MeOH, 7.29 mL, 31.88 mmol) wasadded to a solution of compound 18 (1.00 g, 2.12 mmol) in ethyl formate(5.13 mL, 63.78 mmol) at 0° C. After stirring for 1.5 h, t-BuOMe (10 mL)and 12 N (aq.) HCl (2.66 mL, 31.92 mmol) were added sequentially. Afterstirring for another 5 min, the reaction mixture was transferred to areparatory funnel, which was extracted with EtOAc. The combined extractswere washed with water. The organic layer was separated, which was driedover MgSO₄, filtered, and concentrated. The crude product was mixed withNH₂OH—HCl (0.22 g, 3.17 mmol), water (2 mL) and EtOH (35 mL). Thereaction mixture was heated at 65° C. for 3.5 h, after which EtOH wasremoved by evaporation. The residue was partitioned between EtOAc andwater. The organic extract was separated, which was dried over MgSO₄,filtered, and evaporated. The residue was purified by silica gelchromatography (0% to 60% EtOAc in hexanes) to give compound 19 (820 mg,78% yield) as a white solid: m/z 496.3 (M+1).

Compound 20: Oxalyl chloride (110 μL, 1.30 mmol) was added to a solutionof compound 19 (195 mg, 0.39 mmol) in CH₂Cl₂ (4 mL) at 0° C., followedby the addition of catalytic amount of DMF. The reaction was stirred atroom temperature for 2 h, after which CH₂Cl₂ was evaporated under vacuumto give acid chloride as a light yellow foam solid.

Et₃N (113 μL, 0.81 mmol) and a solution of acethydrazide (50 mg, 0.67mmol) in CH₂Cl₂ (2 mL) were added sequentially to a suspension of theacid chloride in ether (4 mL) at 0° C. The reaction was warmed to roomtemperature and stirred for 30 min. EtOAc was then added, and the crudemixture was transferred to a separatory funnel, which was washed withwater, 1N (aq.) HCl, water. The organic layer was separated, which wasdried over MgSO₄, filtered, and concentrated. The crude product waspurified by column chromatography (silica gel, 0% to 100% EtOAc inhexanes) to give product 20 (215 mg, 99% yield) as a white foam solid:¹H NMR (400 MHz, CDCl₃) δ 8.14 (d, 1H, J=5.2 Hz), 8.05 (d, 1H, J=5.2Hz), 8.00 (s, 1H), 2.86 (m, 2H), 2.34 (m, 3H), 2.09 (s, 3H), 1.80-2.18(m, 8H), 1.34-1.74 (m, 7H), 1.33 (s, 3H), 1.23 (s, 3H), 1.16-1.26 (m,2H), 1.08 (s, 3H), 1.00 (s, 6H), 0.93 (s, 3H), 0.84 (s, 3H).

Compound 21 and Compound 22: A suspension of compound 20 (215 mg, 0.39mmol) and Lawesson's reagent (190 mg, 0.47 mmol) in toluene was heatedat reflux for 30 min. After cooling to room temperature, the reactionmixture was purified by column chromatography (silica gel, 0% to 65%EtOAc in hexanes) to give product 21 (21 mg, 10% yield) as a lightyellow foam solid: m/z 550.3 (M+1). From the column, compound 22 (60 mg,29% yield) was also obtained as a white foam solid: m/z 534.3 (M+1).

Compound 23: NaOMe (25 w/w % solution in MeOH, 17 μL, 0.074 mmol) wasadded to a solution of compound 21 (33 mg, 0.060 mmol) in MeOH (0.6 mL)at room temperature. The reaction was then heated to 55° C., and stirredfor 1 h. After cooling to 0° C., t-BuOMe and 1 N (aq.) HCl were added,and stirred for 5 min. The reaction mixture was transferred to aseparatory funnel, which was extracted with EtOAc. The combined EtOAcextracts were washed with water, dried over MgSO₄, filtered, andconcentrated. The crude product was purified by column chromatography(silica gel, 0% to 45% EtOAc in hexanes) to give product 23 (24 mg, 73%yield) as a white foam solid: m/z 550.3 (M+1). Compound 23 is anisomeric mixture of ketone and enol forms.

Compound 63274: To a solution of compound 23 (23 mg, 0.041 mmol) in DMF(0.3 mL) was added 1,3-dibromo-5,5-dimethylhydantion (6.1 mg, 0.021mmol) at 0° C., and the reaction was stirred at 0° C. for 1 h. Pyridine(14 μL, 0.17 mmol) was then added, and the mixture was heated at 55° C.for 3 h. After cooling to room temperature, the reaction was dilutedwith EtOAc, and transferred to a reparatory funnel, which was thenwashed with Na₂SO₃ (aq.) solution and water. The organic layer wasseparated, which was dried over MgSO₄, filtered, and concentrated. Thecrude product was purified by column chromatography (silica gel, 0% to45% EtOAc in hexanes) to give product 63274 (18 mg, 79% yield) as awhite foam solid: ¹H NMR (400 MHz, CDCl₃) δ 7.61 (s, 1H), 2.97 (d, 1H,J=4.4 Hz), 2.89 (m, 1H), 2.74 (s, 3H), 2.42 (dd, 1H, J=4.8, 16.4 Hz),2.29 (dd, 1H, J=13.6, 16.4 Hz), 2.29 (m, 1H), 2.02 (m, 1H), 1.94 (dd,1H, J=4.8, 13.2 Hz), 1.77-1.91 (m, 3H), 1.52-1.66 (m, 4H), 1.36-1.50 (m,4H), 1.26 (m, 1H), 1.19 (s, 3H), 1.13 (m, 1H), 1.11 (s, 3H), 1.09 (s,3H), 1.02 (s, 3H), 1.00 (s, 3H), 0.96 (s, 3H), 0.80 (s, 3H); m/z 548.3(M+1).

Compound 24: LiAlH₄ solution (1.0 M in THF, 42 mL, 42 mmol) was added toa solution of compound 1 (5.0 g, 10.3 mmol) in THF (100 mL) at roomtemperature under N₂. After stirring for 20 min at room temperature,LiAlH₄ solution (1.0 M in THF, 21 mL, 21 mmol) was added again and thereaction mixture was refluxed for 1 h. After cooling to 0° C., water (10mL) was added dropwise, followed by the addition of 1N HCl (aq) (300mL). The mixture was extracted with EtOAc. The combined extracts werewashed with water, dried with MgSO₄, and concentrated. The residueobtained was mixed with CH₂Cl₂ (200 mL). The white solid thatprecipitated was collected by filtration and washed with CH₂Cl₂ (2×100mL) to give compound 24 (500 mg, 10%) as a white solid. The combinedfiltrate was loaded on a silica gel column and eluted with 0% to 100%EtOAc in hexanes to give additional compound 24 (800 mg, 17%) as a whitesolid: ¹H NMR (400 MHz, CDCl₃) δ 3.79 (m, 1H), 3.54 (m, 2H), 3.20 (dd,1H, J=4.8, 10.8 Hz), 1.98 (m, 1H), 1.12-1.88 (m, 23H), 1.03 (s, 3H),0.98 (s, 6H), 0.91 (s, 3H), 0.86 (s, 3H), 0.85 (s, 3H), 0.77 (s, 3H),0.65-1.10 (m, 3H); m/z 443.3 (M−H₂O+1), 425.3 (100%, M−2×H₂O+1).

Compound 25: TEMPO (27 mg×4, 0.17 mmol×4) and IPh(OAc)₂ (563 mg×4, 1.74mmol×4) were added to a white slurry of compound 24 (725 mg, 1.59 mmol)in CH₂Cl₂ (200 mL) and water (0.1 mL) at 0 h, 2 h, 24 h and 48 h at roomtemperature. After stirring at room temperature for 72 h (overallreaction time), the reaction mixture turned into a clear pink solution,which was then transferred to a separatory funnel and washed with Na₂SO₃(aq) solution. The organic phase was separated, dried over MgSO₄,filtered, and evaporated. The residue was purified by silica gelchromatography (0% to 75% EtOAc in hexanes) to give compound 25 (560 mg,77%) as a white solid: ¹H NMR (400 MHz, CDCl₃) δ 9.37 (d, 1H, J=1.2 Hz),3.77 (m, 1H), 3.18 (dd, 1H, J=4.8, 11.2 Hz), 2.51 (m, 1H), 0.98-1.87 (m,23H), 0.97 (s, 3H), 0.96 (s, 3H), 0.94 (s, 3H), 0.92 (m, 1H), 0.90 (s,3H), 0.86 (s, 3H), 0.82 (s, 3H), 0.75 (s, 3H), 0.65 (m, 1H); m/z 441.3(M−H₂O+1), 423.3 (M−2×H₂O+1).

Compound 26: To a stirred suspension of (Ph₃PCH₂Cl)Cl (4.224 g, 12.1mmol) in THF (13 mL) was added a solution of n-BuLi (4.8 mL, 11.64 mmol,2.5 M in Hexanes) dropwise within 5 minutes at 0° C., followed by theaddition of HMPA (2.4 mL). The reaction was stirred at r.t. for 20minutes and then Compound 25 (1.332 g, 2.90 mmol) in THF (13.0 mL) wasadded within 1 minute. The reaction mixture was stirred at roomtemperature for 2 h, then quenched with HCl (1N, 20 mL) and extractedwith EtOAc (100 mL). The organic phase was washed by HCl (1N, 10 mL),NaCl (Sat., 20 mL), dried over Na₂SO₄, filtered, and concentrated. Thecrude product was purified by column chromatography (silica gel, 0% to10% to 30% EtOAc in hexanes) to give compound 26 (1.2508 g, 87.8%, amixture of E/Z isomers) as a white solid:

Compound 27: To a stirred solution of 26 (1.2508 g, 2.55 mmol) in THF(17 mL) was added a solution of MeLi (5.16 mL, 15.44 mmol, 3 M inCH₂(OEt)₂) dropwise within 1 minute at 0° C. The mixture was thenstirred at room temperature for 28 h and quenched with HCl (1N, 15 mL).The aqueous solution was extracted with EtOAc (2×100 mL). The combinedorganic phase was washed with water, NaCl (sat.), dried over Na₂SO₄,filtered, and concentrated to give compound 27 (1.0630 g, 91.7%) as awhite solid: m/z 437.3 (M—OH).

Compound 28: To a stirred mixture of 27 (881.7 mg, 1.94 mmol), NaOAc(628.6 mg, 4 eq.) in CH₂Cl₂ (40 mL) was added PCC (1.257 g, 3 eq.) inone-portion at room temperature. The mixture was then stirred at roomtemperature for 5 h and diluted with a solvent mixture of EtOAc/Hexanes(1:1, 50 mL). The mixture was directly loaded on a silica gel pad, whichwas then eluted throughout with a solvent mixture of EtOAc/Hexanes(1:1). The eluate was collected and concentrated to give a colorlesscrystalline product. This crude product was purified by columnchromatography (silica gel, 0% to 10% to 25% EtOAc in hexanes) to givecompound 28 (685 mg, 78.8%) as a white solid: m/z 451.3 (M+1).

Compound 29: To a stirred suspension of 28 (22.0 mg, 0.0488 mmol) inHCO₂Et (0.118 mL, 1.46 mmol) was added a solution of MeONa (0.167 mL,0.732 mmol, 25% w/w in MeOH) at 0° C. The mixture was then stirred atroom temperature for 25 h, diluted with TBME (1.4 mL) and quenched withHCl (0.126 mL, conc.) followed by water (3 mL). The aqueous solution wasextracted with EtOAc (10 mL). The combined organic phase was washed withbrine (5 mL), dried over Na₂SO₄, filtered, and concentrated to givecompound 29 as a light yellow foam, which was used directly in the nextstep.

Compound 30: Compound 29 was dissolved in EtOH (2.1 mL). To thissolution were added NH₂OH.HCl (5.1 mg, 0.0732 mmol) and H₂O (0.27 mL) atroom temperature. The mixture was heated at 60° C. for 18 h and thencooled to room temperature. The organic volatiles were removed in vacuo.The remaining mixture was extracted with EtOAc (10 mL). The organicphase was washed with water, brine, dried over Na₂SO₄, filtered, andconcentrated. The crude product was purified by column chromatography(silica gel, 0% to 10% to 25% EtOAc in hexanes) to give 30 (20.8 mg,89.6% from 6) as a colorless crystalline solid: m/z 476.3 (M+1).

Compound 31: To a stirred suspension of 30 (20.8 mg, 0.0437 mmol) in asolvent mixture of MeOH (0.66 mL) and THF (0.11 mL) was added a solutionof MeONa (23.8 μL, 0.105 mmol, 25% w/w in MeOH) at 55° C. The mixturewas then stirred at 55° C. for 3 h, cooled to room temperature andquenched with 1N HCl (aq) (5 mL). The mixture was extracted by EtOAc (15mL). The organic phase was washed with brine, dried over Na₂SO₄,filtered, and concentrated to give compound 31 as a light yellow foam:m/z 476.3 (M−17).

Compound 63303: Compound 31 was dissolved in benzene (2 mL). To thissolution was added a solution of DDQ (10.4 mg, 0.0458 mmol) in benzene(1 mL) at 85° C. The mixture was stirred at 85° C. for 1.5 h, cooled toroom temperature, and quenched with sat. NaHCO₃ (aq) (5 mL). The mixturewas extracted with EtOAc (30 mL). The organic phase was washed with sat.NaHCO₃ (aq) and brine, then was dried over Na₂SO₄, filtered, andconcentrated to give a solid residue (a mixture of starting material anddesired product), which was then dissolved in pyridine (0.5 mL). To thissolution were added Ac₂O (50 μL) and DMAP (cat.) at room temperature.The mixture was stirred at room temperature for 30 min and then quenchedwith NaHCO₃ (sat.). The mixture was extracted with EtOAc (20 mL). Theorganic phase was washed with NaHCO₃ (sat.), HCl (1N), brine, dried overNa₂SO₄, filtered, and concentrated to give a crude mixture, which waspurified by column chromatography (silica gel, 0% to 10% to 25% EtOAc inhexanes) to give 63303 (8.1 mg, 39.1% from 30) as a colorless solid: ¹HNMR (400 MHz, CDCl₃) δ 7.66 (s, 1H), 3.25 (d, 1H, J=4.0 Hz), 2.25-2.52(m, 3H), 2.22 (s, 1H), 1.78-2.15 (m, 5H), 1.44-1.76 (m, 9H), 1.08-1.36(m, 2H), 1.29 (s, 3H), 1.23 (s, 3H), 1.19 (s, 3H), 1.17 (s, 3H), 0.89(s, 3H), 0.94 (s, 3H), 0.91 (s, 3H); m/z 474.3 (M+1).

Compound 32: Compound 31 (95 mg, 0.2 mmol) was dissolved in a solventmixture of acetone (3.5 mL) and water (1.5 mL). To this solution wereadded HgSO₄ (5.9 mg, 0.02 mmol) and H₂SO₄ (2 drops, conc.) at roomtemperature. The mixture was stirred at 55° C. for 20 h, cooled to roomtemperature, and quenched with water (20 mL) and 1 N HCl(aq) (10 mL).The mixture was extracted with EtOAc (30 mL). The organic phase waswashed with 1 N HCl(aq), water, sat. NaHCO₃ (aq), brine, dried overNa₂SO₄, filtered, and concentrated to give a white solid, which waspurified by column chromatography (silica gel, 0% to 10% to 25% EtOAc inhexanes) to give compound 32 (90.3 mg, 91.5%) as a white foam: m/z 494.3(M+1).

Compound 63308: The procedure described for the synthesis of product63274 from compound 23 was then employed to convert compound 32 intoproduct TX63308 (36.1 mg, 73.4%) as a white foam: ¹H NMR (400 MHz,CDCl₃) δ 7.65 (s, 1H), 2.75-2.85 (m, 1H), 2.67 (d, 1H, J=4.4 Hz), 2.44(dt, 1H, J=16.4, 4.8 Hz), 2.35 (dt, 1H, J=16.0, 13.2 Hz), 2.16 (s, 3H),1.92-2.06 (m, 3H), 1.30-1.76 (m, 12H), 1.18-1.29 (m, 1H), 1.22 (s, 3H),1.16 (s, 3H), 1.15 (s, 3H), 1.04 (s, 3H), 0.97 (s, 3H), 0.96 (s, 3H),0.93 (s, 3H); m/z 492.3 (M+1).

Compound 63323: 1,8-Diazabicyclo[5,4,0]undec-7-ene (0.18 mL, 1.204 mmol)was added to a suspension of compound 402-51 (402 mg, 0.814 mmol) intoluene (5.4 mL) at room temperature. After stirring for 2 min, benzylbromide (0.12 mL, 1.009 mmol) was added. The reaction mixture was heatedat 100° C. for 6 h, after which it was cooled to room temperature. Thereaction was then diluted with EtOAc, and was transferred to aseparatory funnel, which was washed with 1 N HCl(aq) and brine. Theorganic extracts were separated, which was dried over Na₂SO₄, filteredand concentrated. The crude product was purified by columnchromatography (silica gel, 0% to 20% EtOAc in hexanes) to give product63323 (308 mg, 65% yield) as a pale yellow foam solid: ¹H NMR (400 MHz,CDCl₃) δ 7.56 (s, 1H), 7.30-7.37 (m, 5H), 5.21 (d, 1H, J=12.4 Hz), 5.07(d, 1H, J=12.4 Hz), 2.84 (m, 1H), 2.47 (d, 1H, J=4.0 Hz), 2.36 (dd, 1H,J=4.4, 16.0 Hz), 2.17 (dd, 1H, J=13.6, 16.0 Hz), 1.81-1.92 (m, 4H),1.20-1.72 (m, 12H), 1.19 (s, 3H), 1.12 (s, 3H), 1.07 (s, 3H), 0.99 (s,3H), 0.90 (s, 6H), 0.65 (s, 3H); m/z 584.4 (M+1).

Compound 63325: MeONH₂—HCl (109 mg, 1.305 mmol), water (0.4 mL) and Et₃N(0.24 mL, 1.722 mmol) were added sequentially to a solution of compound3 (439 mg, 0.857 mmol) in THF (4.2 mL) at room temperature. The reactionwas then heated at 40° C. for 4 h. After cooling to room temperature,the reaction was diluted with EtOAc, and was transferred to a separatoryfunnel, which was washed with 1 N HCl(aq) and brine. The organicextracts were separated, which was dried over Na₂SO₄, filtered andconcentrated. The crude product was purified by column chromatography(silica gel, 10% to 75% EtOAc in hexanes) to give product 63325 (165 mg,37% yield) as a white solid: ¹H NMR (400 MHz, CDCl₃) δ 8.37 (s, 1H),7.63 (s, 1H), 3.76 (s, 3H), 2.86 (d, 1H, J=4.0 Hz), 2.71 (m, 1H), 2.44(dd, 1H, J=4.8, 16.4 Hz), 2.34 (dd, 1H, J=13.2, 16.4 Hz), 1.91-2.08 (m,3H), 1.74-1.90 (m, 2H), 1.59-1.68 (m, 3H), 1.40-1.50 (m, 4H), 1.21 (s,3H), 1.18-1.38 (m, 4H), 1.15 (s, 3H), 1.14 (s, 3H), 1.12 (s, 3H), 0.97(s, 3H), 0.96 (s, 3H), 0.91 (s, 3H); m/z 523.4 (M+1).

Compound 63326: Me₂NH (2.0 M solution in THF, 1.23 mL, 2.460 mmol) wasadded to a solution of compound 3 (408 mg, 0.797 mmol) in THF (4.1 mL)at room temperature. The reaction was then heated at 40° C. for 71 h.After cooling to room temperature, the reaction was diluted with EtOAc,and was transferred to a separatory funnel, which was washed with 1 NHCl(aq) and brine. The organic extracts were separated, which was driedover Na₂SO₄, filtered and concentrated. The crude product was purifiedby column chromatography (silica gel, 10% to 70% EtOAc in hexanes) togive product 63326 (254 mg, 61% yield) as a white solid: ¹H NMR (400MHz, CDCl₃) δ 7.64 (s, 1H), 3.06 (s, 6H), 2.97 (m, 1H), 2.29-2.42 (m,2H), 1.94-2.06 (m, 3H), 1.74-1.85 (m, 2H), 1.61-1.67 (m, 5H), 1.24-1.54(m, 6H), 1.20 (s, 3H), 1.14 (s, 3H), 1.13 (s, 3H), 1.10 (m, 1H), 1.05(s, 3H), 0.99 (s, 3H), 0.96 (s, 3H), 0.91 (s, 3H); m/z 521.4 (M+1).

Compound 33: NH₂OH—HCl (705 mg, 10.145 mmol), NaOAc (1.169 mg, 14.251mmol) and water (3.3 mL) were added to a suspension of compound 402-49(520 mg, 1.020 mmol) in EtOH (9.8 mL) at room temperature. The reactionmixture was heated at 80° C. for 27 h, after which it was cooled to roomtemperature. The reaction mixture was transferred to a separatoryfunnel, which was extracted with EtOAc. The combined organic extractswere washed with water and brine, then dried over Na₂SO₄, filtered andconcentrated. The crude product was purified by column chromatography(silica gel, 0% to 20% EtOAc in hexanes) to give product 33 (387 mg, 72%yield) as a white solid: m/z 525.3 (M+1).

Compound 34: NaOMe (25 w/w % solution in MeOH, 0.12 mL, 0.525 mmol) wasadded to a solution of compound 33 (128 mg, 0.244 mmol) in MeOH (1.2 mL)at room temperature. The reaction was then heated to 55° C. and stirredfor 1 h. After cooling to room temperature, the reaction was dilutedwith t-BuOMe (3 mL) and was cooled to 0° C. 1 N HCl (aq) (5 mL) wasadded. After stirring for another 5 min, the reaction mixture wastransferred to a separatory funnel, which was extracted with EtOAc. Thecombined EtOAc extracts were washed with water, dried over Na₂SO₄,filtered, and concentrated to give product 34 (131 mg) as a white solid:m/z 525.3 (M+1). Compound 34 is an isomeric mixture of C3 ketone andenol forms.

Compound 63295: 1,3-Dibromo-5,5-dimethylhydantoin (40 mg, 0.140 mmol) inDMF (0.5 mL) was added to a solution of compound 34 (126 mg, 0.240 mmol)in DMF (1.6 mL) at 0° C. After stirring at 0° C. for 40 min, thereaction was treated with pyridine (40 μL, 0.495 mmol), and was heatedat 55° C. for 7 h. After cooling to room temperature, brine was added,and the reaction mixture was transferred to a separatory funnel, whichwas extracted with EtOAc. The combined organic extracts were washed withbrine, 10% Na₂SO₃(aq) solution, 1 N HCl(aq), and water. The organiclayer was separated, which was dried over Na₂SO₄, filtered, andconcentrated. The crude product was purified by column chromatography(silica gel, 0% to 20% EtOAc in hexanes) to give product 63295 (34 mg,27% yield from 33) as a white solid: ¹H NMR (400 MHz, CDCl₃) δ 7.83 (s,1H), 3.68 (s, 3H), 3.36 (dd, 1H, J=16.8, 4.8 Hz), 2.82-2.91 (m, 1H),2.54 (d, 1H, J=3.6 Hz), 1.76-2.06 (m, 4H), 1.52-1.74 (m, 6H), 1.04-1.50(m, 8H), 1.20 (s, 3H), 1.15 (s, 3H), 1.13 (s, 3H), 0.93 (s, 3H), 0.92(s, 6H), 0.90 (s, 3H); m/z 523.3 (M+1);

Compound 35: POCl₃ (0.14 mL, 1.502 mmol) was added to a solution ofcompound 33 (200 mg, 0.381 mmol) in pyridine (1.9 mL) at roomtemperature. After stirring for 5 h, the reaction mixture was dilutedwith EtOAc (5 mL), and was quenched with 1 N HCl(aq) (5 mL). Thereaction mixture was transferred to a separatory funnel, which wasextracted with EtOAc. The combined organic extracts were washed with 1 NHCl(aq) and brine, then dried over Na₂SO₄, filtered, and concentrated.The crude product was purified by column chromatography (silica gel, 0%to 50% EtOAc in hexanes) to give product 35 (185 mg, 75% yield) as acolorless glassy solid: m/z 525.4 (M+1);

Compound 36: NaOMe (25 w/w % solution in MeOH, 0.17 mL, 0.743 mmol) wasadded to a solution of compound 35 (177 mg, 0.337 mmol) in MeOH (1.7 mL)at room temperature. The reaction was then heated to 55° C. and stirredfor 4 h. After cooling to 0° C., t-BuOMe and 1 N HCl(aq) were added, andthe reaction mixture was stirred for 5 min. The reaction mixture wasthen transferred to a separatory funnel, which was extracted with EtOAc.The combined EtOAc extracts were washed with 1 N HCl(aq) and brine, thendried over Na₂SO₄, filtered, and concentrated to give product 36 (164mg, 93% yield) as a white solid: m/z 525.4 (M+1). Compound 36 is anisomeric mixture of C3 ketone and enol forms.

Compound 63296: 1,3-Dibromo-5,5-dimethylhydantion (53 mg, 0.185 mmol) inDMF (0.8 mL) was added to a solution of compound 36 (163 mg, 0.311 mmol)in DMF (2.1 mL) at 0° C. After stirring at 0° C. for 1 h, the reactionwas treated with pyridine (50 μL, 0.618 mmol) and was heated at 55° C.for 23 h. After cooling to room temperature, brine was added, and thereaction mixture was transferred to a separatory funnel, which wasextracted with EtOAc. The combined organic extracts were washed withbrine, 10% Na₂SO₃(aq) solution, 1 N HCl(aq), and water, then dried overNa₂SO₄, filtered, and concentrated to give product 63296 (150 mg, 93%yield) as a white solid: ¹H NMR (400 MHz, CDCl₃) δ 7.88 (s, 1H), 5.57(d, 1H, J=4.8 Hz), 4.06 (t, 1H, J=6.0 Hz), 3.73 (s, 3H), 2.68 (dd, 1H,J=14.4, 10.4 Hz), 2.48-2.60 (m, 1H), 2.13 (d, 1H, J=14.4 Hz), 1.65-1.87(m, 3H), 1.19-1.64 (m, 13H), 1.17 (s, 3H), 1.13 (s, 3H), 1.11 (s, 3H),1.06 (s, 3H), 1.00 (s, 3H), 0.97 (s, 3H), 0.88 (s, 3H); m/z 523.3 (M+1).

Compound 37: m-CPBA (77%, 7.04 g, 31.52 mmol) was added to a solution ofcompound 402-49 (1.60 g, 3.15 mmol) in CH₂Cl₂ (28 mL) at roomtemperature. After stirring for 8 h, additional m-CPBA (77%, 3.52 g,15.71 mmol) was added, and the reaction was stirred for another 40 h.Na₂SO₃ (aq.) solution was then added. After another 10 min, the reactionmixture was transferred to a separatory funnel, which was extracted withEtOAc. The combined EtOAc extracts were washed with NaHCO₃ (aq.)solution, dried over MgSO₄, filtered, and concentrated. The crudeproduct was purified by column chromatography (silica gel, 0% to 45%EtOAc in hexanes) to give product 37 (358 mg, 22% yield) as a white foamsolid: m/z 526.3 (M+1).

Compound 38: NaOMe (25 w/w % solution in MeOH, 20 μL, 0.087 mmol) wasadded to a solution of compound 37 (38 mg, 0.072 mmol) in MeOH (0.7 mL)at room temperature. The reaction was then heated to 55° C., and stirredfor 2 h. After cooling to 0° C., t-BuOMe and 1 N (aq.) HCl were added.The reaction mixture was then transferred to a separatory funnel, whichwas extracted with EtOAc. The combined EtOAc extracts were washed withwater, dried over MgSO₄, filtered, and concentrated. The crude productwas purified by column chromatography (silica gel, 0% to 50% EtOAc inhexanes) to give product 38 (25 mg, 66% yield) as a white foam solid:m/z 526.4 (M+1). Compound 38 is an isomeric mixture of C3 ketone andenol forms.

Compound 63263: A solution of 1,3-dibromo-5,5-dimethylhydantion (6.9 mg,0.024 mmol) in DMF (0.2 mL) was added to a solution of compound 38 (25mg, 0.048 mmol) in DMF (0.8 mL) at 0° C. After stirring at 0° C. for 1h, the reaction was treated with pyridine (12 μL, 0.15 mmol), and washeated at 55° C. for 3 h. After cooling to room temperature, thereaction was diluted with EtOAc, and transferred to a separatory funnel,which was then washed with Na₂SO₃ (aq.) solution, 1N (aq.) HCl andwater. The organic extract was separated, dried over MgSO₄, filtered,and concentrated. The crude product was purified by columnchromatography (silica gel, 0% to 45% EtOAc in hexanes) to give product63263 (18 mg) as a white foam solid: ¹H NMR (400 MHz, CDCl₃) δ 7.83 (s,1H), 4.95 (d, 1H, J=7.2 Hz), 3.73 (s, 3H), 2.79 (m, 2H), 2.37 (d, 1H,J=14.4 Hz), 1.92 (m, 2H), 1.77 (d, 1H, J=10.4 Hz), 1.44-1.74 (m, 8H),1.20-1.41 (m, 5H), 1.19 (s, 3H), 1.15 (s, 3H), 1.13 (s, 3H), 1.08 (s,3H), 1.07 (s, 3H), 0.96 (s, 3H), 0.89 (s, 3H); m/z 524.3 (M+1).

Compound 39: LiAlH₄ (2.0 M in THF, 48 μL, 0.096 mmol) was added to asolution of compound 37 (50 mg, 0.095 mmol) in THF (0.95 mL) at 0° C.After stirring for 40 min, the reaction was quenched by adding water (1mL) carefully. After stirring at room temperature for 10 min, thereaction mixture was transferred to a separatory funnel, which wasextracted with EtOAc. The combined organic extracts were washed with 1N(aq.) HCl, and water, dried over MgSO₄, filtered, and concentrated. Thecrude product was purified by column chromatography (silica gel, 0% to40% EtOAc in hexanes) to give product 39 (31 mg, 62% yield) as a whitefoam solid: m/z 528.3 (M+1). The stereochemical configuration of C12 wasnot assigned.

Compound 40: NaOMe (25 w/w % solution in MeOH, 16 μL, 0.070 mmol) wasadded to a solution of compound 39 (30 mg, 0.057 mmol) in MeOH (0.6 mL)at room temperature. The reaction was then heated to 55° C., and stirredfor 2 h. After cooling to 0° C., t-BuOMe and 1 N (aq.) HCl were added.The reaction mixture was then transferred to a separatory funnel, whichwas extracted with EtOAc. The combined EtOAc extracts were washed withwater, dried over MgSO₄, filtered, and concentrated. The crude productwas purified by column chromatography (silica gel, 0% to 40% EtOAc inhexanes) to give product 40 (25 mg, 83% yield) as a white foam solid:m/z 510.3 (M−18+1). Compound 40 is an isomeric mixture of C3 ketone andenol forms. The stereochemical configuration of C12 was not assigned.

Compound 63289: 1,3-Dibromo-5,5-dimethylhydantion (6.8 mg, 0.024 mmol)was added to a solution of compound 40 (25 mg, 0.047 mmol) in DMF (0.47mL) at 0° C. After stirring at 0° C. for 1 h, the reaction was treatedwith pyridine (12 μL, 0.15 mmol), and was heated at 55° C. for 3 h.After cooling to room temperature, the reaction was diluted with EtOAc,and transferred to a separatory funnel, which was then washed withNa₂SO₃ (aq.) solution, 1N (aq.) HCl and water. The organic extract wasseparated, dried over MgSO₄, filtered, and concentrated. The crudeproduct was purified by column chromatography (silica gel, 0% to 40%EtOAc in hexanes) to give partially purified product 63289 (16 mg),which was purified again by preparative TLC plate (silica gel, elutedwith 8% EtOAc in hexanes) to give product 63289 (10 mg, 40% yield) as awhite foam solid: ¹H NMR (400 MHz, CDCl₃) δ 7.64 (s, 1H), 5.12 (m, 1H),4.33 (d, 1H, J=6.8 Hz), 3.71 (s, 3H), 2.54 (m, 1H), 2.43 (d, 1H, J=2.8Hz), 1.21-1.96 (m, 18H), 1.19 (s, 6H), 1.13 (s, 3H), 1.05 (s, 3H), 1.02(s, 3H), 0.95 (s, 3H), 0.88 (s, 3H); m/z 508.3 (M−18+1). Thestereochemical configuration of C12 was not assigned.

Example 4 Uptake of 404-02 into CNS and Lungs of Monkeys

Plasma concentrations following oral dosing: Compound 404-02 shows highuptake in the CNS and lung in monkeys following oral dosing: 2 male and2 female cynomolgus monkeys were administered 404-02 at 0.5, 5, 25 or 75mg/kg/day doses via oral gavage. Doses were prepared in sesame oil andindividualized to body weight on the day of dosing. Blood was drawnprior to dosing and at 1, 2, 4, 8 and 24 hours post-dose on days 1 and12. Blood samples were collected from the femoral artery/vein fordetermination of 404-02 plasma concentrations. Blood was placed in tubescontaining K3EDTA and stored on ice until centrifugation at roomtemperature. The isolated plasma was transferred to cryovials and storedat −80° C. until sample processing and LC-MS/MS analysis. Extractedplasma standard curves were prepared from fresh stock solutions andanalyzed prior to study samples. Summary results are shown in Tables 2aand 2b.

Population mean pharmacokinetic parameter estimates were obtained byperforming non-compartmental analysis of the 404-02 plasmaconcentration-versus-time data using WinNonlin™ software version 5.2.Across the investigated dose range, 404-02 demonstrated dose-dependentkinetics with increased oral clearance (Cl/F), reciprocal shortening ofelimination half-life (T_(1/2)) and an increasing apparent volume ofdistribution (V_(z)/F) with increasing dose. A 1.6-fold increase in thearea under the concentration versus time curve over 24 hours(AUC_(0-24hr)) was observed at the 75 mg/kg dose level after 12 days ofdosing compared to the corresponding AUC on day 1. Accumulation was notobserved at any of the other dose levels. The observed mean maximumplasma concentration (C_(max)) for the 0.5, 5, 25 and 75 mg/kg/day dosegroups on day 12 were 4.6, 12.7, 17.5 and 48.6 nM 404-02, respectively.

Table 2a shows the population mean plasma pharmacokinetics of 404-02 incynomolgus monkey on day 1 of study (n=4). Pharmacokinetic parametersobtained using non-compartmental analysis, WinNonlin™ version 5.2.

TABLE 2a Day 1 Plasma Pharmacokinetics of 404-02: Mean Maximum 404-02Mean Maximum 404-02 Dose Plasma Concentration Plasma Concentration (nM)± (mg/kg/d) (ng/mL) ± SEM SEM 0.5  1.95 ± 0.35  3.4 ± 0.61 5 10.1 ± 3.817.6 ± 6.6 25 16.8 ± 1.3 29.3 ± 2.3 75 19.4 ± 2.7 33.8 ± 4.7

Table 2b shows the population mean plasma pharmacokinetics of 404-02 incynomolgus monkey on day 12 of study (n=4). Pharmacokinetic parameterswere obtained using non-compartmental analysis using WinNonlin™ version5.2 software.

TABLE 2b Day 12 Plasma Pharmacokinetics of 404-02: Mean Maximum 404-02Mean Maximum 404-02 Dose Plasma Concentration Plasma Concentration (nM)± (mg/kg/d) (ng/mL) ± SEM SEM 0.5 2.65 ± 0.6  4.6 ± 1.0 5  7.3 ± 1.712.7 ± 2.9 25 10.0 ± 3.7 17.5 ± 6.4 75  28.0 ± 12.2  48.6 ± 12.2

CNS and lung concentrations following oral dosing: 2 male and 2 femalecynomolgus monkeys were administered 404-02 at 0.5, 5, 25 or 75mg/kg/day doses via oral gavage, in addition to a control(non-treatment) group with 2 animals per sex. Animals were sacrificedapproximately 3 hours after dosing on day 15 following harvest of brainand lung tissues. Each sample collected was rinsed in 1× isotonicphosphate buffered saline and blotted dry before weighing. Harvestedtissue slices were transferred to cryovials and stored at −80° C. untilprocessing and LC-MS/MS analysis. Standard curves were derived for404-02 in homogenates of these tissues and were used to quantify the day15 samples.

The concentration of 404-02 necessary for 50% suppression of nitricoxide (NO) production in macrophages stimulated with interferon-gamma isapproximately 45 nM. As evidenced by the data, 0.5 mg/kg administeredorally as the lowest dose in this study for 15 days resulted in a mean404-02 CNS concentration of 2,162 nM, which markedly exceeds the IC₅₀value for NO production in vitro. The CNS penetration provides a largetherapeutic margin at all doses tested (Table 2c). For instance, themean 404-02 CNS concentrations following administration of 0.5, 5, 25and 75 mg/kg/day 404-02 indicates a 48-, 44-, 37- and 75-fold excesscompared to the dose necessary to suppress inflammation in vitro. Forcomparison, the mean 404-02 lung tissue exposure at 75 mg/kg/day 404-02showed a 133-fold increase (Table 2d). Non-linear disposition of 404-O₂in CNS and lung tissue was observed suggesting 404-02 is taken up by asaturable mechanism(s) for transport across membranes and/orintracellular binding. In addition, concentrations of 404-02 in monkeyCNS and lung tissue exceed plasma levels. Table 2c shows the populationmean 404-02 cynomolgus monkey CNS tissue exposure on day 15. Table 2dshows the population mean 404-02 cynomolgus monkey lung tissue contenton day 15.

TABLE 2c Day 15 CNS Tissue Content/Concentration of 404-02: Mean 404-02CNS Mean 404-02 CNS Dose Tissue Content Tissue Concentration (mg/kg/day)(ng/g) ± SEM (nM)* ± SEM 0.5 1198 ± 712  2087 ± 1239 5 1090 ± 564 1900 ±982 25  930 ± 352 1618 ± 612 75 1898 ± 496 3305 ± 864 *Conversion basedon the assumption the density of tissue is equal to water, 1 g/mL.

TABLE 2d Day 15 Lung Tissue Content/Concentration of 404-02: Mean 404-02Lung Mean 404-02 Lung Dose Level Tissue Content Tissue Concentration(mg/kg/day) (ng/g) ± SEM (nM)* ± SEM 0.5 705 ± 292 1227 ± 508  5 24 ± 1555 ± 27 25 141 ± 50  246 ± 87  75 3405 ± 824  5929 ± 1435

Example 5 Uptake of 404-02 into CNS and Lungs of Rats

Compound 404-02 reaches high concentrations in the lung and CNS of ratsfollowing oral dosing: To assess basic pharmacokinetic parametersfollowing oral dosing, 9 male and 9 female Sprague Dawley (SD) rats wereadministered 404-02 at 1, 10 or 50 mg/kg doses via oral gavage. Doseswere prepared in sesame oil and individualized to body weight on the dayof dosing. Blood was drawn at 0, 1, 2, 4, 8 and 24 hours post-dose ondays 1 and 15. Blood was collected from the orbital sinus after carbondioxide/oxygen inhalation for determination of 404-02 plasmaconcentrations. Plasma was transferred to cryovials and stored at −80°C. until processing and LC-MS/MS analysis. Summary results for Day 1 andDay 15 are shown in Tables 2a and 2b, respectively. A standard curve wasderived for 404-02 in rat plasma, and quantification of experimentalresults was based on this standard curve.

Table 3a shows population mean plasma pharmacokinetics of 404-02 in SDrats on day 1 of study (n=9/sex/dose level). Pharmacokinetic parameterswere obtained using non-compartmental analysis, WinNonlin™ version 5.2.

TABLE 3a Day 1 Pharmacokinetic Parameters Mean Maximum 404-02 MeanMaximum 404-02 Dose Plasma Concentration Plasma Concentration (nM) ±(mg/kg/day) (ng/mL) ± SEM SEM 1 3.3 ± 0.65  5.7 ± 1.1 10  66 ± 16.5  116 ± 28.7 50 713 ± 98.2  1241 ± 171

Table 3b shows the population mean plasma pharmacokinetics of 404-02 inSD rats on day 15 of study (n=9/sex/dose level). Pharmacokineticparameters were obtained using non-compartmental analysis, WinNonlin™version 5.2.

TABLE 3b Day 15 Pharmacokinetic Parameters: Mean Maximum 404-02 MeanMaximum 404-02 Dose Plasma Concentration Plasma Concentration (nM) ±(mg/kg/day) (ng/mL) ± SEM SEM 1 6.3 ± 0.9 11.0 ± 1.6 10  144 ± 24.3  210 ± 42.3 50 1129 ± 114  1966 ± 199

To examine tissue concentrations following oral dosing, 5 male and 5female Sprague Dawley (SD) rats were administered 404-02 at 1, 10, 50 or150 mg/kg/day via oral gavage. Doses were prepared in sesame oil andindividualized to body weight on the day of dosing. Animals weresacrificed 3 hours post-dose on day 15 of study following harvest ofbrain and lung specimens. Each sample collected was rinsed in 1×isotonic phosphate buffered saline and blotted dry before weighing.Tissue slices were transferred to cryovials and stored at −80° C. untilprocessing and LC-MS/MS analysis. Summary results for CNS and lungsamples are shown in Tables 3c and 3d, respectively. Standard curveswere derived for 404-02 in these tissues.

TABLE 3c Population Mean 404-02 CNS Tissue Content in SD Rats on Day 15.Mean 404-02 CNS Tissue Dose Content (ng/g = ng/mL)* ± Mean 404-02 CNSTissue (mg/kg/day) SEM Concentration (nM) ± SEM 1   29 ± 6.8   51 ± 11.810 421 ± 99  733 ± 173 50  750 ± 108 1306 ± 188 150 640 ± 82 1114 ± 143*Conversion based on the assumption the density of tissue is equal towater, 1 g/mL.

TABLE 3d Population Mean 404-02 Lung Tissue Content in SD Rats on Day15. Mean dh404 Lung Tissue Dose Content (ng/g = ng/mL)* ± Mean dh404Lung Tissue (mg/kg/day) SEM Concentration (nM) ± SEM 1 558 ± 123 972 ±215 10 4032 ± 928  7020 ± 1616 50 7165 ± 1221 12474 ± 2126  150 9719 ±1643 16921 ± 2860  *Conversion based on the assumption the density oftissue is equal to water, 1 g/mL.

Example 6 Rodent Toxicity Comparison Between 402 and 402-02

A study was performed in Sprague Dawley rats using both 402 and 402-02.Animals were dosed orally once daily for 7 days. The low dose 402 grouphad elevated total bilirubin and GGT levels as well as suppressed weightgain. The high dose animals that were treated with 402 were allsacrificed in extremis on Day 6 before study completion. GGT and totalbilirubin levels were elevated in these animals as well. However, notoxicity as assessed by clinical observations, weight gain, GGT, andtotal bilirubin was observed in any animal treated with 402-02 (Table4). In a second study involving oral administration to Sprague-Dawleyrats for 14 days, 402-02, achieved comparable blood levels to that of402. However, no significant toxicity was observed as assessed by weightloss, clinical observations, and GGT and total bilirubin elevationsrelative to controls at doses up to 1,500 mg/m²/day for 14 days, whichis 50-fold higher than the MTD of RTA 402 in this species (Table 2).

TABLE 4 Comparing Compounds 402-02 and 402 for Rodent Toxicity WeightTotal vs GGT Bilirubin Timepoint/Dose Level Survival Control (U/L)(mg/dL) 7 Day Study - Crystalline Forms of 402 and 402-02 VehicleControl 4/4 (100%) 100% <5 0.13 402 - 60 mg/m²/day 4/4 (100%)  35% 8.70.25 402 - 180 mg/m²/day 0/4 (0%)  NA 5.0 0.78 402-02 - 60 mg/m²/day 4/4(100%) 106% <5 0.20 402-02 - 180 mg/m²/day 4/4 (100%) 132% <5 0.20 14Day Study - Crystalline Form of 402-02 Vehicle Control 10/10 (100%) 100% <5 0.20 402-02 - 60 mg/m²/day 10/10 (100%)   95% <5 0.24 402-02 -180 mg/m²/day 5/5 (100%) 102% <5 0.22 402-02 - 600 mg/m²/day 5/5 (100%) 96% <5 0.20 402-02 - 1,500 5/5 (100%) 105% <5 0.20 mg/m²/day

Example 7 Toxicity Comparison in Mice

In this study, six compounds (401, 402, 404, 401-2, 402-2, and 404-2)were assessed for toxicity in mice in a 14-day study. Each compound wasformulated in sesame oil and administered daily by oral gavage at dosesof 10, 50, 100, or 250 mg/kg (n=4 per group). At higher doses (above 10mg/kg/day) both 401 and 402 caused at least 50% mortality; 402 wasnon-toxic. In contrast, no mortality was observed in the 402-2 and 404-2groups and only the highest dose of 401-02 caused any lethality (Table5). Body weight measurements (FIGS. 29-31) were consistent with themortality observations. The two highest doses of 401 and 402 were lethalwithin 4 days, in contrast to the effects of 401-2 and 402-2.

TABLE 5 Mortality Observations in 14-Day Toxicity Study Number Dose ofGroup Compound (mg/kg) Schedule N Deaths Comments  1 vehicle QD×14, 4 0D1-14  2 401 10 QD×14, 4 0 D1-14  3 401 50 QD×14, 4 2 D1-14  4 401 100QD×14, 4 4 D1-14  5 401 250 QD×14, 4 4 D1-14  6 401-02 10 QD×14, 4 0D1-14  7 401-02 50 QD×14, 4  1* *Due to D1-14 gavage injury  8 401-02100 QD×14, 4 0 D1-14  9 401-02 250 QD×14, 4 1 Sacrificed D1-14 due toweightless on Day 11 10 402 10 QD×14, 4 0 D1-14 11 402 50 QD×14, 4 4D1-14 12 402 100 QD×14, 4 4 D1-14 13 402 250 QD×14, 4 4 D1-14 14 402-0210 QD×14, 4 0 D1-14 15 402-02 50 QD×14, 4 0 ID1-14 15 402-02 100 QD×14,4 0 D1-14 17 402-02 250 QD×14, 4 0 D1-14  1a 404 10 QD×14, 4 0 D1-14 19404 50 QD×14, 4 0 D1-14 20 404 100 QD×14, 4 0 D1-14 21 404 250 QD×14, 40 D1-14 22 404-02 10 QD×14, 4 0 D1-14 23 404-02 50 QD×14, 4 0 D1-14 22404-02 100 QD×14, 4 0 D1-14 23 404-02 250 QD×14, 4 0 D1-14

In a second experiment, six additional compound differing only in thesaturation or non-saturation of the C ring were tested for toxicity inmice by daily oral administration for 9 days, using sesame oil as thevehicle. In this study, no significant toxicity was observed. The deathsof two animals were attributed to gavage errors during theadministration of the test article. No significant differences in weightwere observed in any group compared to the vehicle-treated controls.Results are summarized in Table 6 below. As with compounds 402-2, 401-2and 404-2 above, compounds with saturation in the C ring consistentlyshow low toxicity in rodents. Compounds lacking saturation in the C ringshow significant rodent toxicity in some cases (e.g., 401 and 402).Predictably low rodent toxicity provides an advantage since high rodenttoxicity can be a significant complication in conducting preclinicalstudies required for development and registration of therapeuticcompounds for use in humans or non-human animals.

TABLE 6 Further Mouse Toxicity Results. Compound Dose (per day, p.o.)Mortality 63112  3 mg/kg 0/5 10 mg/kg 0/5 30 mg/kg 1/5 63323  3 mg/kg0/5 10 mg/kg 0/5 30 mg/kg 0/5 63324  3 mg/kg 0/5 10 mg/kg 0/5 30 mg/kg0/5 63325  3 mg/kg 0/5 10 mg/kg 0/5 30 mg/kg 0/5 63166  3 mg/kg 0/5 10mg/kg 0/5 30 mg/kg 0/5 63326  3 mg/kg 0/5 10 mg/kg 1/5 30 mg/kg 0/5

Example 8 Aqueous Solubility of Oleanolic Acid Derivatives

The aqueous solubility of the compounds shown here was determined usingthe procedures outlined in Example 1.

Aqueous Solubility Compound ID(s) Structure (μM) 63097 (402)

1.46 63102 (dh404)

0.06 63198

163.6 63202

1.89 63208

9.49 63214

112.2 63219

13.58 63221

8.78 63226

0.71 63231

1.23 63232

0.75 63237

5.16

All of the methods disclosed and claimed herein can be made and executedwithout undue experimentation in light of the present disclosure. Whilethe compositions and methods of this invention have been described interms of preferred embodiments, it will be apparent to those of skill inthe art that variations may be applied to the methods and in the stepsor in the sequence of steps of the method described herein withoutdeparting from the concept, spirit and scope of the invention. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, and those listed in the Appendix, to theextent that they provide exemplary procedural or other detailssupplementary to those set forth herein, are specifically incorporatedherein by reference.

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What is claimed is:
 1. A pharmaceutical composition comprising acompound of the formula:

wherein: Y is cyano, heteroaryl_((C≦12)), substitutedheteroaryl_((C≦12)), or —C(O)R_(a), further wherein: R_(a) is: hydrogen,hydroxy, halo, amino, azido, mercapto or silyl; or alkyl_((C≦12)),alkenyl_((C≦12)), alkynyl_((C≦12)), aryl_((C≦12)), aralkyl_((C≦12)),heteroaryl_((C≦12)), heteroaralkyl_((C≦12)), alkoxy_((C≦12)),alkenyloxy_((C≦12)), alkynyloxy_((C≦12)), aryloxy_((C≦12)),aralkoxy_((C≦12)), heteroaryloxy_((C≦12)), heteroaralkoxy_((C≦12)),acyloxy_((C≦12)), alkylamino_((C≦12)), alkoxyamino_((C≦12)),dialkylamino_((C≦12)), alkenylamino_((C≦12)), alkynylamino_((C≦12)),arylamino_((C≦12)), aralkylamino_((C≦12)), heteroarylamino_((C≦12)),heteroaralkylamino_((C≦12)), amido_((C≦12)), or a substituted version ofany of these groups; and X is OR_(b), or NR_(b)R_(c), wherein R_(b) andR_(c) are each independently: hydrogen or hydroxy; alkyl_((C≦8)),aryl_((C≦8)), aralkyl_((C≦8)), acyl_((C≦8)), alkoxy_((C≦8)),aryloxy_((C≦8)), acyloxy_((C≦8)), alkylamino_((C≦8)), arylamino_((C≦8)),amido_((C≦8)), or a substituted version of any of these groups; orprovided that R_(b) is absent when the atom to which it is bound is partof a double bond, further provided that when R_(b) is absent the atom towhich it is bound is part of a double bond; or a pharmaceuticallyacceptable salt, tautomer, or optical isomer thereof; and apharmaceutically acceptable carrier.
 2. The pharmaceutical compositionof claim 1, wherein the compound is further defined as:

wherein R_(a) is: hydrogen, hydroxy, halo or amino; or alkyl_((C≦8)),alkenyl_((C≦8)), alkynyl_((C≦8)), aryl_((C≦8)), aralkyl_((C≦8)),heteroaryl_((C≦8)), heteroaralkyl_((C≦8)), alkoxy_((C≦8)),alkenyloxy_((C≦8)), alkynyloxy_((C≦8)), aryloxy_((C≦8)),aralkoxy_((C≦8)), heteroaryloxy_((C≦8)), heteroaralkoxy_((C≦8)),acyloxy_((C≦8)), alkylamino_((C≦8)), alkoxyamino_((C≦8)),dialkylamino_((C≦8)), alkenylamino_((C≦8)), alkynylamino_((C≦8)),arylamino_((C≦8)), aralkylamino_((C≦8)), heteroarylamino_((C≦8)),heteroaralkylamino_((C≦8)), amido_((C≦8)), or a substituted version ofany of these groups; or a pharmaceutically acceptable salt, tautomer, oroptical isomer thereof.
 3. The pharmaceutical composition of claim 2,wherein the compound is further defined as:

wherein R_(a) is alkoxy_((C1-4)), alkylamino_((C1-4)),alkoxyamino_((C1-4)), dialkylamino_((C2-4)), or a substituted version orany of these groups; or a pharmaceutically acceptable salt, tautomer, oroptical isomer thereof.
 4. The pharmaceutical composition of claim 2,wherein the compound is further defined as:

wherein R_(a) is hydrogen, hydroxy, amino, methoxy, or2,2,2-trifluoroethylamino; or a pharmaceutically acceptable salt,tautomer, or optical isomer thereof.
 5. The pharmaceutical compositionof claim 1, wherein the compound is further defined as:

wherein Y is heteroaryl_((C≦8)) or a substituted heteroaryl_((C≦8)); ora pharmaceutically acceptable salt, tautomer, or optical isomer thereof.6. The pharmaceutical composition of claim 1, wherein X is OR_(b). 7.The pharmaceutical composition of claim 6, wherein R_(b) is absent. 8.The pharmaceutical composition of claim 6, wherein R_(b) is hydrogen. 9.The pharmaceutical composition of claim 1, wherein X is NR_(b).
 10. Thepharmaceutical composition of claim 9, wherein R_(b) is hydroxy.
 11. Thepharmaceutical composition of claim 1, wherein the compound is furtherdefined as:


12. The pharmaceutical composition of claim 1, wherein the compound isfurther defined as:


13. The pharmaceutical composition of claim 1, wherein the compound isfurther defined as:


14. The pharmaceutical composition of claim 1, wherein the compound isfurther defined as:


15. The composition of claim 1, wherein the compound is further definedas:


16. The pharmaceutical composition of claim 1, wherein the compound isfurther defined as:


17. The pharmaceutical composition of claim 1, wherein the compound isfurther defined as:


18. The pharmaceutical composition of claim 1, wherein the compound isfurther defined as:


19. The pharmaceutical composition of claim 1, wherein the compound isfurther defined as:


20. The pharmaceutical composition of claim 1, wherein the compound isfurther defined as:


21. The pharmaceutical composition of claim 1, wherein the compound isfurther defined as:


22. The pharmaceutical composition of claim 1, wherein the compound isfurther defined as:


23. The pharmaceutical composition of claim 1, wherein the compositionis formulated for oral delivery.
 24. A pharmaceutical compositioncomprising a compound of the formula:

and a pharmaceutically acceptable carrier.